25.1: DNA-Dependent Synthesis of RNA

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Learning Goals 

(Learning goals written by Claude, Sonnet 4.6, Anthropic)

RNA Classes and the RNA Polymerase Machinery

• Distinguish the major classes of RNA by size, structure, and function: explain how coding mRNA differs from noncoding RNAs including short ncRNAs (miRNA, siRNA; ~20–30 nt), small ncRNAs (tRNA, snRNA, snoRNA; up to 200 nt), and long ncRNAs (rRNA, lncRNA, eRNA; >200 nt)—and explain why rRNA dominates cellular RNA by mass (~90%) while mRNA and lncRNA are present at 1–2 orders of magnitude lower levels, with lncRNA showing highly cell-type-specific expression patterns despite producing more than 100,000 distinct species.
• Compare prokaryotic and eukaryotic RNA polymerases: explain how the bacterial RNAP core (α₂ββ'ω) and its sigma factor (forming the holoenzyme) correspond structurally to the conserved 10-subunit core found across eukaryotic Pol I, II, and III; describe how the three eukaryotic RNAPs divide transcriptional labor (Pol I → most rRNA; Pol II → all mRNA and most regulatory ncRNA; Pol III → tRNA and 5S rRNA); and explain how RNAP I and III appear to have integrated transcription factor-like functions into their core structures while Pol II recruits discrete general transcription factors (GTFs) to form the preinitiation complex.

Transcription Initiation: Prokaryotic and Eukaryotic Mechanisms

• Describe bacterial transcription initiation: explain how sigma factor recognizes the −35 and −10 (Pribnow box) promoter elements, how flipping of the A−11 base into its σ2 recognition pocket initiates promoter melting to form the open complex (RPo), how the transcription bubble forms, and how sigma dissociation accompanies the transition to processive elongation—and distinguish how different sigma factors (σ70 for housekeeping genes, σ38 for starvation response) direct the holoenzyme to different gene sets.
• Explain the stepwise assembly of the eukaryotic RNA Pol II preinitiation complex (PIC): describe how TFIID (TBP + 13 TAFs) initiates PIC assembly by binding the TATA box through TBP's phenylalanine residues inserting into the minor groove, causing a sharp bend and partial strand separation; how sequential recruitment of TFIIA, TFIIB, TFIIF, RNA Pol II, TFIIE, and TFIIH forms the closed complex; and how TFIIH's XPB helicase/translocase uses ATP to induce torsional strain, opening ~11–15 bp around the transcription start site to position ssDNA at the Pol II active site and permit initiation.
• Explain the roles of enhancers, the Mediator complex, and promoter-proximal pausing in regulating Pol II transcription: describe how enhancers act over long distances in a position- and orientation-independent manner to drive transcription in bursts, how the Mediator complex physically bridges enhancers to promoters through chromatin looping, and how after synthesizing ~20–100 nt most Pol II complexes pause and must escape the promoter—with only TFIIB, TFIIF, and Pol II needing to be re-recruited for subsequent rounds of re-initiation.

Transcriptional Elongation and Termination

• Describe the molecular mechanism of transcription elongation and backtracking rescue: explain how NTP binding induces trigger loop (TL) folding, followed by phosphodiester bond formation and pyrophosphate release, and RNAP translocation along the template; explain how backtracking (caused by thermodynamic properties of local sequences or misincorporation) displaces the 3′ RNA end from the active site; and describe how the intrinsic hydrolytic activity of the TL provides slow rescue, while GreA/GreB factors in prokaryotes (or their eukaryotic analogs) bind the secondary channel, displace the TL, and catalyze rapid RNA cleavage by stabilizing the second Mg²⁺ ion and attacking water molecule to restore a functional elongation complex.
• Compare intrinsic and Rho-dependent transcription termination in prokaryotes: explain how intrinsic termination is driven by formation of an RNA hairpin stem-loop (from an inverted repeat sequence) followed by a run of U residues that destabilizes the weak rU:dA hybrid and causes TEC disassembly; and describe how the Rho hexameric ATPase-helicase binds C-rich Rut sites via its primary binding sites, translocates along the nascent RNA using ATP hydrolysis via conformational changes in Q and R loops of the secondary binding site, and catches up with a paused RNAP to promote TEC disassembly.
• Describe Pol II transcription termination for coding versus noncoding transcripts: explain how mRNA termination requires a functional polyadenylation signal (AAUAAA hexamer) recognized by cleavage and polyadenylation factors (CPF), with termination proceeding through either the allosteric model (pA signal induces conformational change in Pol II leading to TEC disassembly) or the torpedo model (XRN2/Rat1p 5′→3′ exonuclease degrades the downstream nascent transcript after cleavage at the pA site and disassembles the elongating complex); and explain how ncRNA termination in yeast uses Nrd1/Nab3 RNA-binding proteins to recruit the Sen1 helicase and nuclear exosome rather than the polyadenylation machinery.

Structurally speaking, ribonucleic acid (RNA) is quite similar to DNA. However, whereas DNA molecules are typically long and double-stranded, RNA molecules are much shorter and are typically single-stranded. A ribonucleotide within the RNA chain contains ribose (the pentose sugar), one of the four nitrogenous bases (A, U, G, and C), and a phosphate group. The subtle structural difference between the sugars gives DNA added stability, making it more suitable for storing genetic information. In contrast, the relative instability of RNA makes it more suitable for its short-term functions. The RNA-specific pyrimidine uracil forms a complementary base pair with adenine and is used instead of thymine, which is found in DNA. Even though RNA is single-stranded, most types of RNA molecules show extensive intramolecular base pairing between complementary sequences within the RNA strand, creating a predictable three-dimensional structure essential for their function, as shown in Figure \(\PageIndex{1}\) and Figure \(\PageIndex{2}\).

Figure \(\PageIndex{1}\): RNA Structural Elements (a) Ribonucleotides contain the pentose sugar ribose instead of the deoxyribose found in deoxyribonucleotides. (b) RNA contains the pyrimidine uracil in place of thymine found in DNA. Figure from: Parker et al (2019) Microbiology from Openstax

Figure \(\PageIndex{2}\): Parker, et al (2019) Microbiology from Openstax

RNA can be primarily divided into two types: one that carries the code for making proteins, known as coding RNA, also referred to as messenger RNA (mRNA), and non-coding RNA (ncRNA). ncRNAs can be subdivided into several types, depending on either their length or their function. Size classification begins with the short ncRNAs (~20–30 nt), which include microRNAs (miRs), and small interfering (siRNAs); the small ncRNAs up to 200 nt, which include transfer RNA (tRNA), small nuclear RNA (snRNA), and small nucleolar RNA (snoRNA); and long ncRNAs ( > 200 nt), which include ribosomal RNA (rRNA), enhancer RNA (eRNA) and long intergenic ncRNAs (lincRNAs), among others.

Cells access the information stored in DNA by creating RNA through transcription, which then directs protein synthesis through translation. The three main types of RNA directly involved in protein synthesis are messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA). mRNA carries the message from DNA, which controls all cellular activities within a cell. If a cell requires a certain protein to be synthesized, the gene for this protein is “turned on,” and the mRNA is synthesized through the process of transcription. The mRNA then interacts with ribosomes and other cellular machinery to direct the synthesis of the protein it encodes during translation. mRNA is relatively unstable and short-lived in the cell, especially in prokaryotic cells, ensuring that proteins are only made when needed.

rRNA and tRNA are stable types of RNA. In prokaryotes and eukaryotes, tRNA and rRNA are encoded by DNA and transcribed into long RNA molecules that are subsequently processed to yield smaller fragments containing the individual mature RNA species. In eukaryotes, synthesis, cutting, and assembly of rRNA into ribosomes take place in the nucleolus region of the nucleus, but these activities occur in the cytoplasm of prokaryotes. Within the nucleolus region, ribosome assembly requires the activity of numerous snoRNAs.

Ribosomes are composed of rRNA and protein. As its name suggests, rRNA is a major constituent of ribosomes, accounting for approximately 60% of the ribosome's mass and providing the site where mRNA binds. The rRNA ensures the proper alignment of mRNA, tRNA, and proteins. The rRNA of the ribosome also has enzymatic activity (peptidyl transferase) and catalyzes the formation of peptide bonds between two aligned amino acids during protein synthesis (Figure 26.1.3). Although rRNA had long been thought to serve a structural role primarily, its catalytic role within the ribosome was shown in 2000. Scientists in the laboratories of Thomas Steitz (1940–) and Peter Moore (1939–) at Yale University successfully crystallized the ribosome structure from Haloarcula marismortui, a halophilic archaeon isolated from the Dead Sea. Due to the significance of this work, Steitz shared the 2009 Nobel Prize in Chemistry with other scientists who made substantial contributions to the understanding of ribosome structure. The structure and function of ribosomes will be discussed in further detail in Chapter 27.

Transfer RNA (tRNA) is the third prominent type of RNA involved in protein translation. tRNAs are usually only 70–90 nucleotides long. They carry the correct amino acid to the site of protein synthesis in the ribosome. It is the base pairing between the tRNA and mRNA that allows for the correct amino acid to be inserted in the polypeptide chain being synthesized, as shown in Figure \(\PageIndex{3}\). Any mutations in the tRNA or rRNA can result in global problems for the cell, as both are essential for proper protein synthesis.

Figure \(\PageIndex{3}\): A generalized illustration of how mRNA and tRNA are used in protein synthesis within a cell. Figure from: Parker et al (2019) Microbiology from Openstax

As previously described, some RNA molecules possess enzymatic properties and function as ribozymes. Within this chapter, the activity of snRNAs during the process of intron removal from mRNA sequences functions as ribozymes and will be described. Furthermore, a detailed description of the enzymatic features of the ribosome structure will be provided in Chapter 27.

Other small ncRNAs and lncRNA molecules also play a role in regulating transcriptional and translational processes. For example, the post-transcriptional expression levels of many genes can be controlled by RNA interference, in which miRNAs, specific short RNA molecules, pair with mRNA regions and target them for degradation, as shown in Figure \(\PageIndex{4}\). This process is facilitated by protein chaperones known as argonautes. This antisense-based process involves steps that first process the miRNA so that it can base-pair with a region of its target mRNA. Once the base pairing occurs, other proteins direct the mRNA to be destroyed by nucleases. Fire and Mello were awarded the 2006 Nobel Prize in Physiology or Medicine for this discovery.

Figure \(\PageIndex{4}\): Role of Micro RNA (miRNA) in the Inhibition of Eukaryotic mRNA Translation. (1) A protein called Exportin-5 transports a hairpin primary micro RNA (pri-miRNA) out of the nucleus and into the cytoplasm. (2) An enzyme called Dicer (not shown) trims the pri-miRNA and removes the hairpin loop. A group of proteins known as Argonautes forms an miRNA/protein complex. (3) The miRNA/protein complex hydrogen bonds with mRNA based on complementary sequence homology and blocks translation. (4,5) The miRNA/protein complex binding speeds up the breakdown of the poly (A) tail of the mRNA, causing the mRNA to be degraded sooner. Figure modified from: Wikimedia Commons

At steady state, the vast majority of human cellular RNA consists of rRNA (∼90% of total RNA for most cells) as shown in Figure \(\PageIndex{5}\). However, there is less tRNA by mass; its small size results in a higher molar level than rRNA (Figure 26.1.5). Other abundant RNAs, such as mRNA, snRNA, and snoRNAs, are present in aggregate at levels about 1–2 orders of magnitude lower than those of rRNA and tRNA (Figure 26.1.5). Certain small RNAs, such as miRNA and piRNA, can be present at very high levels; however, this appears to be cell-type-dependent. lncRNAs are present at levels that are two orders of magnitude less than total mRNA. Although the estimated number of different types of human lncRNAs may have a very restricted expression pattern, they thus accumulate to higher levels within specific cell types. For example, sequencing of mammalian transcriptomes has revealed that more than 100,000 different lncRNA molecules can be produced, compared with the approximate 20,000 protein-coding genes. The diversity and functions of the transcriptome within biological processes are currently a highly active area of research.

Figure \(\PageIndex{5}\): Estimate of RNA levels in a typical mammalian cell. The proportion of the various classes of RNA in mammalian somatic cells by total mass (A) and by the absolute number of molecules (B). The total number of RNA molecules is estimated at roughly 10⁷ per cell. Other ncRNAs in (A) include snRNA, snoRNA, and miRNA. Note that due to their relatively large sizes, rRNA, mRNA, and lncRNA make up a larger proportion of the mass as compared to the overall number of molecules. Figure from: Palazzo, A. and Lee, E.S. (2015) Frontiers in Genetics 6:2

RNA Polymerases

This chapter focuses on RNA synthesis by DNA-dependent RNA Polymerase Enzymes (RNAPs). These enzymes are essential for transcription and are found in all cells, from bacteria to humans. RNAPs are multi-subunit assemblies; bacteria have five core subunits, α2ββ'ω, with homologs in archaeal and eukaryotic RNAPs. Bacterial RNAPs are the simplest form of RNA polymerases, providing an excellent system for studying how they control transcription. We will discuss a much simpler RNAP from the bacteriophage T7 at the end of this chapter.  It has a single chain and is structurally homologous to the T7 DNA polymerase.  It is also a good example of how RNAPs (as well as DNA poluymerase) are motor proteins!

Prokaryotic RNA Polymerase Enzymes

The RNAP catalytic core within bacteria contains five major subunits (α₂ββ'ω) (see Figure \(\PageIndex{7}\)) below. Positioning this catalytic core onto the correct promoter requires the association of a sixth subunit, called the sigma factor (σ). Within bacteria, multiple sigma factors can associate with the catalytic core of RNAP, directing it to the correct DNA locations where transcription can be initiated. For example, in E. coli, σ70 is the housekeeping sigma factor responsible for transcription of most genes in growing cells. It maintains essential genes and pathways. Other sigma factors are activated under specific environmental conditions, such as σ³⁸, which is activated during starvation or when cells reach the stationary phase. When the sigma subunit associates with the RNAP catalytic core, the holoenzyme is formed. When bound to DNA, the holoenzyme conformation of RNA polymerase (RNAP) can initiate transcription.

Transcription takes place in several stages. First, the RNA polymerase holoenzyme locates and binds to the promoter DNA. At this stage, the RNAP holoenzyme is in the closed conformation (RPc), as shown in Figure \(\PageIndex{6}\). Initial specific binding to the promoter by sigma factors of the holoenzyme sets in motion conformational changes in which the RNAP molecular machine bends and wraps the DNA, with mobile regions of RNAP playing key roles, as shown in Figure \(\PageIndex{6}\). Next, RNAP separates the two strands of DNA and exposes a portion of the template strand. At this point, the DNA and the holoenzyme are said to be in an ‘open promoter complex’ (RP_o), and the section of promoter DNA that is within it is known as a ‘transcription bubble’. Intermediates(I_1-3) between RP_c and RP_o occur.

Figure \(\PageIndex{6}\): Schematic Representation of E. coli Transcriptional Initiation. Closed complexes like RPC, I1,E (early), or I1,L (late) can be significant members of the rapidly equilibrating I1 ensemble. RPo signifies the end of the initiation stage and entry into the elongation phase of RNA synthesis. The α domains are shown in light blue; σ domains are indicated by numbers 1.1, 1.2, 2, 3, and 4. Figure from: Ruff, E.F., et. al. (2015) Biomolecules 5(2):1035-1062.

In bacterial systems, the sigma factor locates the transcriptional start site using key DNA sequence elements located at -35 nucleotides and -10 nucleotides from the transcriptional initiation site, as shown in Panel A of Figure \(\PageIndex{7}\). This region is called the Pribnow box. For RNAP from Thermus aquaticus, the −35 element interacts exclusively with σ₄. The duplex DNA just upstream of the −10 element (−17 to −13) interacts with β′, σ₂, and σ₃ (Panel B). Flipping of the A₋₁₁(nt) base from the duplex DNA into its recognition pocket in σ₂ is thought to be the key event in the initiation of promoter melting and the formation of the transcription bubble (Panel C). Once the transcription bubble has formed and transcription initiates, the sigma subunits dissociate from the complex, and the RNAP catalytic subunit continues elongation on its own.

Figure \(\PageIndex{7}\): Structure of the RNAP Holoenzyme in Thermus aquaticus. (Figure modified from: Bae, B., et.al. (2015) eLife 4:e08504

Panel (A) shows oligonucleotides used for the crystallization of the RNAP holoenzyme in the open conformation. The numbers above denote DNA positions relative to the transcription start site (+1). The −35 and −10 (Pribnow box) elements are shaded yellow, and the extended −10 and discriminator elements are purple. The nontemplate-strand DNA (top strand) is colored dark grey; template-strand DNA (bottom strand), light grey; RNA transcript, red.

Panel (B) shows the overall structure of RNAP holoenzyme in the open conformation bound to the DNA nucleotides. The nucleic acids are shown as CPK spheres and color-coded as in diagram A. Within RNAP, the αI, αII, and ω are shown in grey; β in light cyan; β′ in light pink; Δ1.1σ^A in light orange. The Taq EΔ1.1σ^A (Taq derives from Thermus aquaticus) is shown as a molecular surface, and the forward portion of the RNAP holoenzyme is transparent to reveal the RNAP active site Mg²⁺ (yellow sphere) and the nucleic acids held inside the RNAP active site channel.

Panel (C) Electron density and model for RNAP holoenzyme nucleic acids in the open conformation. Color coding matches diagram A.

Figure \(\PageIndex{8}\) shows an interactive iCn3D model of the T. aquaticus transcription initiation complex containing bubble promoter and RNA (4XLN).

Figure \(\PageIndex{8}\): T. aquaticus transcription initiation complex containing bubble promoter and RNA (4XLN). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...pSXsY6HdMDdELA (long load time but worth the wait)

It is color-coded in a fashion similar to that shown in Figure \(\PageIndex{7}\).

The rendering of the DNA is as follows:

• The non-template (nt-strand) DNA is colored dark grey; spheres
• template (t-strand) DNA, light grey; spheres
• Pribnow box yellow
• discriminator purple

The proteins are shown as surfaces with transparent secondary structures underneath. The color is as follows:

• (αI, αII, ω, light yellow;
• β, light cyan;
• β′, light pink;
• Taq EΔ1.1σ^A, light orange),

The RNA polymerase active site is located at the Mg²⁺ (black sphere) binding site. The nucleic acids are within the RNAP active-site channel.

 

Eukaryotic RNA Polymerase Enzymes

In eukaryotic cells, three RNAPs (I, II, and III) share the task of transcription, the first step in gene expression. RNA Polymerase I (Pol I) is responsible for the synthesis of the majority of rRNA transcripts. In contrast, RNA Polymerase III (Pol III) produces short, structured RNAs such as tRNAs and 5S rRNA. RNA Polymerase II (Pol II) produces all mRNAs and most regulatory and untranslated RNAs.

The three eukaryotic RNA polymerases contain homologs to the five core subunits found in prokaryotic RNAPs. In addition, the eukaryotic Pol I, Pol II, and Pol III have five additional subunits forming a catalytic core that contains 10 subunits, as shown in Figure \(\PageIndex{9a}\). The core has a characteristic crab-claw shape that encloses a central cleft harboring the DNA and has two channels: one for the substrate NTPs and the other for the RNA product. Two ‘pinchers’, called the ‘clamp’ and ‘jaw’, stabilize the DNA at the downstream end and allow the opening and closing of the cleft. For transcription to occur, the enzyme must maintain a transcription bubble with separated DNA strands, facilitate nucleotide addition, translocate along the template, stabilize the DNA:RNA hybrid, and finally allow the DNA strands to reanneal. This is achieved through several conserved elements in the active site, including the fork loop(s), rudder, wall, trigger loop, and bridge helix.

Figure \(\PageIndex{9a}\): Structural Diagram of RNA Polymerase from Thermus thermophilus (PDB205j) with archaeal and eukaryotic homologs. Hein, P.P. and Landick, R. (2010) BMC Biology 8:141

DNA (black) is melting into a transcription bubble, allowing template-strand pairing with RNA (red) in a 9-10 base pair RNA-DNA hybrid. The bridge helix (cyan) and trigger loop/helices (yellow/orange) lie on the downstream side of the active site. The straight arrow indicates the presumed path of the NTP entry. The curved arrow indicates the interconversion of the trigger loop and trigger helices. The RNA polymerase subunits are shown as semi-transparent surfaces with the identities of orthologous subunits in bacteria (α, β, and β', gray, blue, and pink, respectively), archaea (D, L, B, and A), and eukaryotic RNA polymerase II (RPB3, 11, RPB2, RPB1) indicated. The active site Mg²⁺ ions are shown as yellow spheres, and α,β-methylene-ATP in green and red.

Figure \(\PageIndex{9b}\) shows an interactive iCn3D model of T. thermophilus RNAP polymerase elongation complex with the NTP substrate analog (2O5J ).

Figure \(\PageIndex{16}\): T. thermophilus RNAP polymerase elongation complex with the NTP substrate analog (2O5J). (Copyright: author via source). Click the image for a popup or use this external link: https://www.ncbi.nlm.nih.gov/Structu...f66bf6e6e2c175

The dark blue strand is single-stranded RNA, complementary to the magenta DNA strand.  The bridge helix (gray/black cylinder) and the bent trigger loop helices (red cylinders) are labeled.  Two sets of flexible hinges in the bridge helix at 1076‑GA‑1077 and 1080‑GG‑1081, which let the helix bend during the catalytic cycle, are shown in gold spacefill. NTP substrate analog is shown in spacefill.  To view just the nucleic acids, use these commands in iCn3D:

• Analysis, Sequences and Annotations
• Click the Details tab, and click and then control-click the nucleic acid strands to select all of them
• View, Selection

Table \(\PageIndex{1}\) shows RNA polymerase (RNA pol) subunit composition in bacteria, archaea, yeast, and plants (both eukaryotes).

---

Bacteria	Archaea	RNA pol I	RNA pol II	RNA pol III	RNA pol IV (plants)	RNA pol V (plants)
β	Rpo1 (RpoA)	RPA190	RPB1	RPC160	NRPD1	NRPE1
β'	Rpo2 (RpoB)	RPBA135	RPB2	RPC128	NRPD/E2	NRPD/E2
α	Rpo3 (RpoD)	RPAC40	RPB3	RPAC40	RPB3 [1]	RPB3 [1]
α	Rpo11 (RpoL)	RPAC19	RPB11	RPAC19	RPB11	RPB11
ω	Rpo6 (RpoK)	RPB6	RPB6	RPB6	RPB6 [1]	RPB6
	Rpo5 (RpoH)	RPB5	RPB5	RPB5	RPB5 [3]	NRPES5
	Rpb8 (RpoG)*	RPB8	RPB8	RPB8	RPB8 [1]	RPB8 [1]
	Rpo10 (RpoN)	RPB10	RPB10	RPB10	RPB10	RPB10
	Rpo12 (RpoP)	RPB12	RPB12	RPB12	RPB12	RPB12
	Rpo4 (RpoF)	RPA14	RPB4	RPC17	NRPD/E4	NRPD/E4
	Rpo7(RpoE)	RPA43	RPB7	RPC25	NRPD7 [1]	NRPE7
		RPA12	RPB9	RPC11	NRPD9b	RPB9
	Rpo13*
		RPA49		RPC53
		RPA34.5		RPC37
				RPC82
				RPC34
				RPC31

Table \(\PageIndex{1}\): RNA polymerase (RNA pol) subunit composition. Abel, C., Verónica, M., I., G. A. , & Francisco, N. (2017). Subunits Common to RNA Polymerases. In (Ed.), The Yeast Role in Medical Applications. IntechOpen. https://doi.org/10.5772/intechopen.70936. Creative Commons Attribution 3.0 License

Schematic representations of the structure of the eukaryotic RNA pols I, II, and III are shown in Figure \(\PageIndex{10}\).

Figure \(\PageIndex{10}\): Schematic representation of the structure of the RNA pols I, II, and III. Each RNA polymerase common subunit is indicated in grey. The numbers corresponding to each subunit are shown in Subunits Common to RNA Polymerases: Abel et al, ibid.

RNA polymerases must bind to DNA and to a host of transcription factors (TFs) necessary for specific and regulable transcription. (Note: RNAP is not considered a transcription factor.) The comparative structures of RNAP I-III are shown in Figure \(\PageIndex{11}\). The "stalk" is a structural feature found in eukaryotes but not in prokaryotes. The figure focuses mostly on a comparison of RNAP I and RNAP III.

Figure \(\PageIndex{11}\): Comparison of RNAPI, II and III structures and transcription factors. Turowski TW and Boguta M (2021) Specific Features of RNA Polymerases I and III: Structure and Assembly. Front. Mol. Biosci. 8:680090. doi: 10.3389/fmolb.2021.680090. Creative Commons Attribution License (CC BY).

Panel (A) illustrates the general architecture of RNAPII, comprising the catalytic core and stalk. RNAPII core consists of a DNA-binding channel, a catalytic center, and an assembly platform. RNAPII binds multiple transcription factors (TFs). Some TFs are homologous to additional subunits of specialized RNAPs (i.e., TFIIF).

Panel (B) shows the subunit composition of eukaryotic RNAPs. Human nomenclature is shown for comparison. Please note that the C-terminal region of Rpa49 subunit harbors a “tandem winged helix” which is predicted in TFIIE and that human RNAPIII RPC7 subunit is coded by two isoforms α and β. The question mark indicates the name is unconfirmed.

Panel (C) shows the subunit composition of yeast RNAPI.

Panel (D) shows a Model of the RNAPI pre-initiation complex, showing an early intermediate with visible Rrn3 and core factor (CF). TATA-binding protein (TBP) and upstream-associated factor (UAF) are added schematically.

Panel (E) shows the subunit composition of yeast RNAPIII.

Panel (F) shows an atomic model of RNAPIII pre-initiation complex with TFIIIB. The Rpc82/34/31 heterotrimer is involved in initiation and is marked in green, as shown in E. TFIIIC is added schematically. PDB: 5C4X, 5FJ8, 4C3J, 6EU0, and 6TPS

The stalks contain two proteins that are less homologous to the core subunits. These are highlighted in blue in Panel B of Figure \(\PageIndex{11}\). RNAP I and III have additional subunits compared to RNAP II. Overall, RNAP I-III have 14, 12, and 17 subunits, respectively. RNAP I and III appear to have integrated transcription-like factors into their core enzyme. In contrast, RNAP II (which transcribes DNA to form messenger RNA), binds to discrete and separate transcription factors to form a preinitiation complex (PIC)which we will discuss below.

Transcription Factors and the Preinitiation Complex (PIC)

Unlike prokaryotic systems which can initiate the recruitment of RNAP holoenzymes directly onto the DNA promoter regions and mediate the conversion of RNAP to the open conformation, eukaryotic RNA polymerases require a host of additional general transcription factors (GTFs), to enable this process. Here, we will focus on the activation of RNA Polymerase II as an example of the complexity of eukaryotic transcription initiation.

Class II gene transcription in eukaryotes is a tightly regulated, essential process controlled by a highly complex multicomponent machinery. A plethora of proteins, more than a hundred in humans, are organized in very large multiprotein assemblies that include a core of General Transcription Factors (GTFs). The GTFs include the factors TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, RNA polymerase (RNA pol II), as well as a large number of diverse complexes that act as co-activators, co-repressors, chromatin modifiers, and remodelers, as shown in Figure \(\PageIndex{12}\). Class II gene transcription is regulated at various levels: during assembly on chromatin, before and during transcription initiation, throughout elongation and mRNA processing, and at termination. A host of activators and repressors have been reported to regulate transcription, including a central multisubunit complex called the Mediator, which facilitates the recruitment of GTFs and the activation of RNA Pol II. Here, we will focus on the formation of the GTFs that comprise the core preinitiation complex (PIC) during transcriptional activation.

Figure \(\PageIndex{12}\): Transcription Preinitiation Complex (PIC). Gupta, K., et.al. (2016) J Mol Bio 428(12):2581-91.

Class II gene transcription in humans is initiated by the assembly of over a hundred polypeptides on the core promoter of protein-encoding genes, which then leads to mRNA production. A PIC on a core promoter is shown schematically. PIC contains, in addition to promoter DNA, the GTFs (TFIIA, B, D, E, F, and H) and RNA Pol II. PIC assembly is thought to occur in a highly regulated, stepwise fashion, as indicated. TFIID is among the first general transcription factors (GTFs) to bind the core promoter via its TATA-box Binding Protein (TBP) subunit. Nucleosomes at transcription start sites contribute to PIC assembly, mediated by signaling through epigenetic marks on histone tails. The Mediator (not shown) is a further central multiprotein complex identified as a global transcriptional regulator. TATA = TATA-box DNA; BRE^u = B recognition element upstream; BRE^d = B recognition element downstream; Inr = Initiator; DPE = Downstream promoter element. Figure from:

Transcription of RNA pol II-dependent genes is triggered by the regulated assembly of the Preinitiation Complex (PIC). PIC formation starts with the binding of TFIID to the core promoter. TFIID is a large megadalton-sized multiprotein complex with around 20 subunits made up of 14 different polypeptides: the TATA-box binding protein (TBP) and the TBP-associated factors (TAFs) (numbered 1–13), as shown in Figure \(\PageIndex{13}\). Some of the TAF subunits are present in two copies. A key feature of TAFs is the histone fold domain (HFD), which is present in 9 of the 13 TAFs in TFIID. The HFD is a strong protein–protein interaction motif that mediates specific dimerization. The HFD-containing TAFs are organized into discrete heterodimers, except TAF10, which can form dimers with two different TFIID components, TAF3 and TAF8. HFDs and several other structural features of TBP and the TAFs are well conserved between species.

Figure \(\PageIndex{12}\): Human TFIID. Gupta, K., et.al. (2016) J Mol Bio 428(12):2581-91.

TFIID is a large megadalton-sized multiprotein complex comprising about 20 subunits made up of 14 different polypeptides. The constituent proteins of TFIID, TBP and the TAFs, are shown schematically as bars (inset, left). Structured domains are marked and annotated. The presumed stoichiometry of TAFs and TBP in the TFIID holo-complex is given (far left, gray underlaid). TAF10 (in italics) makes a histone fold pair separately with both TAF3 and TAF8. TAFs present in a physiological TFIID core complex extracted from eukaryotic nuclei are labeled in bold. The architecture of the TFIID core complex (EMD-2230), as determined by cryo-EM, is shown (bottom left) in two views related by a 90° rotation (arrows). The holo–TFIID complex is characterized by remarkable structural plasticity. Two conformations, based on cryo-EM data (EMD-2284 and EMD-2287), are shown on the right: a canonical form (top) and a more recently observed rearranged form (bottom). In the rearranged conformation, lobe A (colored in red) migrates from one end of the TFIID complex (attached to lobe C) to the other extremity (attached to lobe B).

TFIID was shown to adopt an asymmetric, horseshoe shape with three almost equal-sized lobes (A, B, and C), exhibiting a considerable degree of conformational flexibility with at least two distinct conformations (open and closed), as shown in Figure \(\PageIndex{12}\). The TBP component of TFIID binds with a specific DNA sequence called the TATA box. This DNA sequence is found around 30 base pairs upstream of the transcription start site in many eukaryotic gene promoters. When TBP binds to a TATA box within the DNA, it distorts the DNA by inserting amino acid side chains between base pairs, partially unwinding the helix, and creating a double kink. The distortion is achieved through extensive surface contact between the protein and DNA. TBP binds to the negatively charged phosphates in the DNA backbone through positively charged lysine and arginine residues. The sharp bend in the DNA is produced through the projection of four bulky phenylalanine residues into the minor groove. As the DNA bends, its contact with TBP increases, thus enhancing the DNA-protein interaction. The strain imposed on the DNA through this interaction initiates the melting, or separation, of the strands. Because this region of DNA is rich in adenine and thymine residues, which base-pair through only two hydrogen bonds, the DNA strands are more easily separated.

The role of TAFs is complicated. Take, for example, TAF11 and TAF13. These act as competitive inhibitors of TBP at the TATA (Pribnow) box, as does TAF1, which somewhat mimics the structural features of the Pribnow box. TAF11/TAF13 bind to the DNA surface where TBP binds, suggesting a novel regulation of TFIID. These interactions are illustrated in Figure \(\PageIndex{14}\).

Figure \(\PageIndex{14}\): Novel TFIID regulatory state comprising TAF11/TAF13/TBP. Kapil Gupta, et al. (2017) Architecture of TAF11/TAF13/TBP complex suggests novel regulation properties of general transcription factor TFIID eLife 6:e30395. https://doi.org/10.7554/eLife.30395. Creative Commons Attribution License

Given its role in transcribing DNA to produce thousands of messenger RNAs, let's focus on the preinitiation complex for RNA polymerase II. Structures are known for the closed and open promoters. A key component is TFIIH (see Figure \(\PageIndex{12}\)). TFIIH opens the DNA for transcription. As if we need to complicate the structure of the preinitiation complex even further, it turns out that TFIIH is not a single entity, as depicted in Figure 12, but rather a large complex in its own right. Its structure is shown in Figure \(\PageIndex{15}\).

Figure \(\PageIndex{15}\): Structure of the TFIIH core complex. Greber et al. (2019). The complete structure of the human TFIIH core complex. eLife 8:e44771. https://doi.org/10.7554/eLife.44771. Creative Commons Attribution License

Panes A, B, and C show three views of the structure of the TFIIH core complex and MAT1. Subunits are color-coded and labeled in color; individual domains are labeled in black and circled if necessary for clarity.

Panel (D) shows the domain-level protein-protein interaction network between the components of the TFIIH core complex and MAT1 derived from the interactions observed in our structure. Proteins are shown with the same colors as in A, and major unmodeled regions are shown in grey. Abbreviations: CTD: C-terminal domain; DRD: DNA damage recognition domain; FeS: iron-sulfur cluster domain; NTD: N-terminal domain; vWFA: von Willebrand Factor A.

The largest subunits are DNA-dependent helicases/translocases/ATPases XPB and XPD, which MAT1 bridges. It appears that XPB initiates and propagates a DNA twist, which in turn opens the DNA 30 base pairs (bp) downstream of the TATA box. This is most likely followed by the dissociation of TFIIH and a cessation in DNA twisting, which enables RNA transcription to commence.

Figure \(\PageIndex{16}\) shows an interactive iCn3D model of the human/mouse/mastadenovirus C RNA polymerase II core pre-initiation complex with open promoter DNA (7NVU).

Figure \(\PageIndex{16}\): Human/mouse/a mastadenovirus C RNA polymerase II core pre-initiation complex with open promoter DNA (7NVU). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...Bc8itsYA7qctWA (long load time but worth the wait)

The following color schemes are used:

• MAT1 - orange
• TF2A - yellow
• TF2B - green
• TF2E - magenta
• TF2F- purple
• ATP-dependent translocase (helicase) subunit XPD - dark slate gray
• TBP (TATA box binding protein) - red
• TF2H - Pink
• 2H-XPB helicase - maroon
• NT-DNA - cyan
• template (T)-DNA - blue (Note: part of it is shown in a yellow cartoon in the interactive model)
• the SF₄ cofactor is shown in spacefill with CPK colors

In summary, the binding of TFIID to the core promoter is followed by the recruitment of further GTFs and RNA pol II. Several lines of evidence suggest that this process occurs in a defined, stepwise order and undergoes significant restructuring. First, PIC adopts an inactive state, the “closed” complex, which is incompetent to initiate transcription. In addition to TFIID, TFIIH is also critical for the shift of RNA Pol II from the closed to the open conformation. TFIIH has an ATP-dependent translocase activity within one of its subunits, which opens up about 11 to 15 base pairs around the transcription start site by moving along one DNA strand, inducing torsional strain and leading to conformational rearrangements, thereby positioning single-stranded DNA at the active site of RNA pol II. In this open complex, RNA polymerase II can enter elongation to transcribe through a gene in a highly processive manner without dissociating from the DNA template or losing the nascent RNA.

In most eukaryotes, after synthesizing approximately 20–100 bases, RNA polymerase II (RNA pol II) can pause (promoter-proximal pause) and then dissociate from promoter elements and other components of the transcription machinery, giving rise to a fully functional elongation complex in a process called promoter escape. The promoter-bound components of the PIC, in contrast, remain in place; thus, only TFIIB, TFIIF, and RNA polymerase II need to be recruited for re-initiation, significantly increasing the transcription rate in subsequent rounds of transcription. Promoter escape is preceded by an abortive transcription in many systems, where multiple short RNA products of 3 to 10 bases in length are synthesized.

In addition to promoter elements within the DNA, enhancer elements are also important for the initiation of transcription. Promoters are DNA elements that recruit transcription complexes to synthesize both coding and non-coding RNAs. Enhancers are DNA elements that positively regulate transcription at promoters over long distances, in a position- and orientation-independent manner. However, studies have revealed that many enhancers can recruit Pol II and initiate transcription of enhancer RNA (eRNA), thus blurring the functional distinction between enhancers and promoters (Figure 10.13).

Enhancer transcription produces relatively short ncRNA. Furthermore, transcription at enhancers is unstable and often leads to the termination of elongation. In contrast, transcription initiation at most Pol II promoters is stable and results in the production of long mRNAs. Topological studies revealed that enhancers come near target gene promoters during transcription activation. According to current gene activation models, the Mediator complex forms a physical bridge between distant regulatory regions and promoters, thereby promoting looping. Transcription of at least a subset of genes regulated by enhancers occurs in bursts, indicating a discontinuous process of transcription complex recruitment, assembly, and/or conversion to elongation-competent forms. The bursting phenomenon suggests that enhancer/promoter contacts may be transient and infrequent, as shown in Figure \(\PageIndex{17}\).

Figure \(\PageIndex{17}\): RNA polymerase II Enhancer Transfer Model. Gurumurthy, A., et.al. (2018) BioEssays 1800164

The steps involved in the recruitment of Pol II to SEs, assembly into elongation-competent transcription complexes, transcription initiation, elongation, termination, and transfer to target genes are depicted. Transcription factors recruit Mediator and other co-regulators to SEs. Mediator recruits Pol II and assembles a fraction into elongation-competent transcription complexes. Transcription is initiated by phosphorylation of the CTD. Early abortion and transcription termination are conferred by Integrator release Pol II, which is dephosphorylated and transferred to target gene promoters. Super Enhancer Element (SE).

Transcriptional Elongation and Termination

Prokaryotic Transcriptional Elongation

The rate of transcription elongation by E. coli RNAP is not uniform. RNA synthesis is characterized by pauses, some of which may be brief and resolved spontaneously, whereas others may lead to backtracking of the transcription elongation complex (TEC).

Elongation rate and pausing are determined by the template sequence and RNA structure (e.g., stem-loops) and involve at least two components of the RNAP catalytic center: the bridge helix (BH) and the trigger loop (TL). Elongation is proposed to occur in three steps, as shown in Figure \(\PageIndex{18}\). In the first step, the TL folds in response to NTP binding. Mutational analyses indicate that this conformational change in the TL can be rate-limiting and reflects the ability of the incoming NTP to bind to TEC. The second step is NTP incorporation and release of pyrophosphate. The third step involves the translocation of the RNAP along the DNA template so that the next RNA nucleotide can be added to the nascent transcript.

Figure \(\PageIndex{18}\): A model for Transcriptional Elongation. Kireeva, M., et. al. (2012) BMC Biophysics 5(1):11

The trigger loop hinges, bridge helix hinges, and bridge helix bending models are based on molecular dynamics simulations. At the top of the figure, diagrams of the closed TEC, the closed product TEC (after chemistry), and the translocating TEC are shown. DNA is grey; RNA is red; the NTP substrate (or incorporated NMP and pyrophosphate) is blue; the trigger loop (TL) is purple; the bridge helix (BH) is yellow. Interpretations of simulations are shown schematically below. Simulations indicate trigger loop hinges H1 and H2, bridge helix hinges H3 and H4, and bridge helix bend modes B1 (straighter) and B2 (more sharply bent).

Backtracking of TEC may occur after a brief pause in transcription, due to the thermodynamic properties of the nucleic acid sequences surrounding the elongation complex. In addition, misincorporation events render elongation complexes prone to backtracking by at least one base pair. In this case, the rescue from backtracking through the cleavage of the 3' end of the erroneous transcript may also be seen as a proofreading reaction. Any backtracking event causes a pause or arrest of transcription elongation, which may limit the overall rate (the average speed of RNAP along the template) or processivity (the fraction of RNAP molecules that reach the end of the gene).

While the general structure of the elongation complex, including the transcription bubble and the RNA-DNA hybrid, remains unchanged during backtracking, the extension of RNA becomes impossible in this conformation. However, such complexes can be resolved by the hydrolytic activity of RNAP, which cleaves the phosphodiester bond in the active center of the backtracked complex, producing a new RNA 3' end in the active center. For single-base backups, the hydrolytic reaction is catalyzed by a flexible domain of RNAP located in the secondary channel, known as the Trigger Loop (TL), and the two metal ions of the active center.

Longer backtracked TEC sequences can restart when acted upon by GreA/B factors, which restore the 3'-end of the nascent transcript to the active center. GreA and GreB are transcript cleavage factors that act on backtracked elongation complexes. When Gre factors are bound in the secondary channel, Gre factors displace the TL from the active center, as shown in Figure \(\PageIndex{19}\). The displacement switches off the relatively slow TL-dependent intrinsic transcript hydrolysis and imposes the highly efficient Gre-assisted hydrolysis. This efficiency is thought to be due to Gre factors stabilizing the second catalytic Mg²⁺ ion and an attacking water molecule.

Figure \(\PageIndex{19}\): The Role of Gre Factors in Relieving Transcription Elongation Complex Backtracking. Zenkin, N., and Yuzenkova, Y. (2015) Biomolecules 5(3):1195-1209.

Panel (A) shows a ribbon diagram of the GreA and GreB proteins.

Panel (B) shows the mode of functioning of Gre factors. The Gre factor is bound to the active elongation complex but does not impose hydrolytic activity on it. Upon backtracking or misincorporation, the Gre factor protrudes its coiled-coil domain through the secondary channel of RNAP (as shown in the left-hand diagram), where it substitutes for the catalytic domain's Trigger Loop (TL). This substitution switches off the slow TL-dependent phosphodiester bond hydrolysis and, instead, facilitates highly efficient Gre-dependent hydrolysis. After the resolution of the backtracked complex through RNA cleavage, the elongation complex returns to the active conformation, and the Gre factor gives way to the TL, which can now continue the catalysis of RNA synthesis (shown in the right-hand diagram). The controlled switching between Gre and the TL eliminates potential interference by Gre with RNA synthesis.

Prokaryotic Transcriptional Termination

Transcription termination determines the ends of transcriptional units by disassembling the transcription elongation complex (TEC), thereby releasing RNA polymerases and nascent transcripts from DNA templates. Failure to terminate transcription leads to readthrough, resulting in wasteful and potentially harmful intergenic transcripts. It can also perturb the expression of downstream genes when the unterminating TEC sweeps transcription initiation complexes off their promoters or collides with RNA polymerases that transcribe opposite strands.

Transcriptional termination in prokaryotes can be template-encoded and factor-independent (intrinsic termination) or require accessory factors, such as Rho, Mfd, and DksA. Intrinsic termination occurs at specific template sequences - an inverted repeat followed by a run of A residues. Termination is driven by the formation of a short stem-loop structure in the nascent RNA chain, as shown in Figure \(\PageIndex{20}\). RNA synthesis arrests, and TEC dissociates at the 7th and 8th U of the run. Formation of the stem-loop dissociates the weak rU:dA hybrid. Stem-loop formation is hindered by upstream complementary RNA sequences that compete with the downstream portion of the stem, as well as by RNA:protein interactions in the RNA exit channel. Intrinsic termination depends critically upon timing. Hairpin folding and transcription of the termination point must be coordinated so that the complete hairpin is formed by the time RNAP transcribes the termination point. The stem size, sequence, and loop length all affect termination efficiency.

The bridge α-helix in the β' subunit borders the active site and may have roles in both catalysis and translocation. Mutations in the YFI motif (β' 772-YFI-774) affect intrinsic termination as well as pausing, fidelity, and translocation of RNAP. One mutation, F773V, abolishes the activity of the λ tR2 intrinsic terminator, although neighboring mutations have little effect on termination. Modeling suggests that this unique phenotype reflects F773's ability to interact with the fork domain of the β subunit.

Figure \(\PageIndex{20}\): Model of Intrinsic Termination. Washburn, R.S., and Gottesman, M.E. (2015) Biomolecules 5(2)1063-1078.

Panel (A) shows the open conformation of the RNAP during transcriptional elongation. RNAP is shown in yellow, the DNA template in blue, and the nascent RNA in red. Key elements of the RNAP RNA exit channel are shown in grey and labeled as indicated.

Panel (B) shows the extension of the nascent RNA through the RNAP exit channel and the potential for forming the RNA hairpin structure when enough length has been achieved.

Panel (C) shows the clamp opening and disintegration of the TEC when the RNA hairpin structure is encountered at the transcriptional bubble.

Figure \(\PageIndex{21}\) shows an interactive iCn3D model of the T. thermophilus RNAP polymerase elongation complex with the NTP substrate analog (2O5J). (long load time)

Figure \(\PageIndex{21}\): T. thermophilus RNAP polymerase elongation complex with the NTP substrate analog (2O5J). (Copyright; author via source). Click the image for a popup or use this external link:https://structure.ncbi.nlm.nih.gov/i...g4RAeYA3FiGDLA (long load time)

 

Transcriptional termination can also depend on accessory factors, such as the Rho protein. The transcription termination factor Rho is an essential protein in E. coli, first identified for its role in transcription termination at Rho-dependent terminators. It is estimated to terminate approximately 20% of E. coli transcripts. The rho gene is highly conserved and nearly ubiquitous in bacteria. Rho is an RNA-dependent ATPase with RNA:DNA helicase activity, and consists of a hexamer of six identical monomers arranged in an open circle, as shown in Figure \(\PageIndex{22}\).

Rho binds to single-stranded RNA in a complex multi-step pathway that involves two distinct sites on the hexamer. The primary binding site (PBS), distributed on the N-terminal domains around the hexamer (cyan), ensures initial anchoring of Rho to the transcript at a Rut (Rho utilization) site, a ∼70-nucleotide (nt) long, cytidine-rich, and poorly structured RNA sequence. Each Rho monomer contains a subsite capable of binding specifically the base residues of a 5′-YC dimer (Y being a pyrimidine). Biochemical and structural data suggest that Rho initially binds to RNA in an open, ‘lock-washer’ conformation that closes into a planar ring as RNA transfers to the central cavity. There, the ssRNA contacts an asymmetric secondary binding site (SBS) (green), and this step, which presumably is rate-limiting for the overall reaction, leads to motor activation. Upon ATP hydrolysis, the ssRNA is pulled due to conformational changes in the conserved Q and R loops of the SBS, leading to Rho translocation and ultimately promoting RNA polymerase (RNAP) dissociation. The molecular mechanism of Rho translocation, as determined by single-molecule fluorescence methods, appears to involve tethered tracking. The tethered tracking model postulates that Rho maintains its contacts between the PBS and the loading (Rut) site upon translocation (Panel B). This mechanism would allow Rho to maintain its high-affinity interaction with Rut and would imply the formation of an RNA loop between the PBS and the SBS upon translocation.

Figure \(\PageIndex{22}\): Schematic of Rho factor structure and mechanisms. (A) Jawahar Swaminathan and (B) Gocheva, V., et. al. (2015) Nuc Acids Res 43(1)10.1093.

Panel (A) shows the molecular structure of the Rho protein (PDB 1pv4)

Panel (B)shows how Rho assembles as a homo-hexameric ring (red spheres or tetragons), with RNA (black/yellow curve) binding to the primary binding sites (PBS, cyan) and the secondary binding sites inside the ring (SBS, green), where ATP-coupled translocation takes place. The Rut-specific binding site is depicted in yellow. The tethered-tracking model proposed that Rho translocates RNA while maintaining interactions between PBS and Rut. This model requires the formation of a loop that would shorten the RNA extension upon translocation. Figure modified from:

Figure \(\PageIndex{23}\) shows an interactive iCn3D model of the E. Coli Rho transcription termination factor in complex with ssRNA substrate and ANPPNP (1PVO).

Figure \(\PageIndex{23}\): E. Coli Rho transcription termination factor in complex with ssRNA substrate and ANPPNP (1PVO). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...boj4YBbvevcsEA

The six subunits of the hexamer are shown in alternating slate gray and light gray. The di-ribonucleotides (5'-R(P*UP*C)-3') are shown in spacefill and colored CPK. ANPPNP is shown in spacefill yellow.

Figure \(\PageIndex{24}\) shows an interactive iCn3D model of the closed ring structure of the E. Coli Rho transcription termination factor in a complex with nucleic acid in the motor domains (2HT1).

Figure \(\PageIndex{24}\): Closed ring structure of the E. Coli Rho transcription termination factor in complex with nucleic acid in the motor domains (2HT1). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...GVkVhPknbmiYj7

The six subunits of the hexamer are again shown in alternating slate gray and light gray. The ssRNAs are shown in spacefill with the backbones in one color and the bases in CPK colors.

Figure \(\PageIndex{25}\) shows an interactive iCn3D model of the E. coli Rho-dependent Transcription Pre-termination Complex (6XAS) (long load).

Figure \(\PageIndex{25}\): E. coli Rho-dependent Transcription Pre-termination Complex (6XAS). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...kUxfqN2TFPcSL9 (long load)

• The RNA polymerase subunits (α₂ββω) are shown in light cyan
• The six subunits of the Rho hexamer are again shown in alternating slate gray and light gray
• NusA is shown in magenta
• Both DNA strands are shown in spacefill with the backbone blue and the bases red
• Only part of the RNA is shown (spacefill, backbone yellow, bases CPK), so it appears discontinuous.

 

Eukaryotic Transcriptional Termination

In eukaryotes, the termination of protein-coding gene transcription by RNA polymerase II (Pol II) typically requires a functional polyadenylation (pA) signal, usually a variation of the AAUAAA hexamer. Nascent pre-mRNA is cleaved, and the 5′ fragment is polyadenylated at the pA site shortly downstream from the hexamer by cleavage and pA factors (CPFs). Two mechanisms have been suggested for pA-dependent transcription termination. In the allosteric model, the pA signal and/or other termination signals bind to the pA signal downstream region (PDR), inducing reorganization of the Pol II complex. This includes the association or dissociation of endonuclease components such as the CPFs. This causes conformational changes in Pol II, and TEC disassembly ensues. In the kinetic model, also known as the “torpedo” model, cleavage at the pA site separates the pre-mRNA from the TEC, which continues synthesizing a downstream nascent transcript. This new transcript is a substrate of XRN2/Rat1p, a processive 5′-to-3′ exoribonuclease that catches up with and disassembles the TEC through an unknown mechanism.

The two pA-dependent models are not mutually exclusive, and unified models have been proposed to address this discrepancy. Loosely conserved pA signal sequences downstream of protein-coding genes bind to components of the polyadenylation factor (CF1) complex, leading to the assembly of the cleavage and polyadenylation machinery. Termination is coupled to cleavage in a manner that has not yet been completely resolved. However, one of the major factors involved in yeast pA termination is the endonuclease, Ysh1. For example, the depletion of Ysh1 blocks TEC dissociation, but does not cause substantial readthrough at the termination site (Fig. 26.1.18 A&B). These results suggest that Ysh1 does not directly cause the pausing that occurs in the allosteric termination pathway, but rather plays a role in the dissociation of the Pol II complex from the DNA template, as shown in Figure \(\PageIndex{26}\). It should be noted that not all pA-dependent termination is dependent on Ysh1 and that other mechanisms of pA-mediated termination remain to be elucidated.

Figure \(\PageIndex{26}\): Schematic representation of Pol II termination after removal of non-pA and pA termination factors. Schaughency, P., Merran, J., and Corden J.L. (2014) PLOS Genetics 10(10):e1004632

Panel (A) shows the elongating Pol II (green) terminates pA transcripts (A) after an allosteric change (red) that reduces processivity.

Panel (B) shows that the depletion of Ysh1 leads to minimally extended readthrough transcripts but does not block the allosteric change in Pol II.

Panel (C) shows how Nrd1 and Nab3 binding recruits Sen1 to terminate non-pA transcripts.

Panel (D) shows that the Pol II elongation complex lacking Nrd1 does not recognize termination sequences in the nascent transcript and thus does not facilitate the allosteric transition in Pol II. This leads to a processive readthrough.

Panel(E) shows how Nrd1 and Nab3 recognize terminator sequences, allowing the allosteric change in Pol II, but depletion of Sen1 blocks the removal of Pol II from the template. Figure from:

The mechanisms of Pol II-mediated transcription termination differ between coding and non-coding transcripts. Coding transcripts and possibly some stable uncharacterized transcripts (SUTs) are nearly always processed at the 3′-end by the cleavage and polyadenylation (pA) machinery. They are processed by the pA-dependent termination mechanisms described above. In contrast, ncRNAs are terminated and processed by an alternative pathway that, in yeast, requires the RNA-binding proteins Nrd1 and Nab3, as well as the RNA helicase Sen1. Nrd1 and Nab3 recognize RNA sequence elements downstream of snoRNAs and CUTs, and this leads to the association of a complex that contains the DNA/RNA helicase Sen1 and the nuclear exosome. The nuclear exosome is a complex of ribonucleases with 3' to 5' exonuclease and endonuclease activity. It functions to degrade unstable or incorrect RNA transcripts.

Depletion of both Nrd1 and Sen1 leads to readthrough transcription of ncRNAs, suggesting their importance in non-pA-dependent transcription termination (Fig. 26.1.18 C & D). Furthermore, depletion of Nrd1 also causes the accumulation of longer readthrough ncRNAs, suggesting its role in trafficking ncRNAs to the nuclear exosome following termination.

Recent Updates:  8/24/26

A simple RNA polymerase molecular motor from the bacteriophage T7

Let's turn our attention to a simpler DNA-dependendt RNA polymerase from the bacteriophage T7.  In contrast to the complexity of the eukaryotic and prokaryotic RNAP complexes described above (with multiple subunits and complex regulation), the T7 RNAP is a single-subunit protein without the clamp, bridge helix, or trigger loop found in the bacterial/eukaryotic RNA polymerases (E. coli, Thermus, yeast Pol II).  It has a different fold, similar to DNA polymerase I (Klenow-fold: fingers, palm, thumb, plus an N-terminal domain).  Its biophysical properties are likewise easier to study.  

The N-terminal domain binds the 23-base-pair DNA promoter (without the need for a preannealed primer as in DNA polymerases, including from T7) during initiation using a specificity loop (amino acids 740-769) and a helix-loop-helix motif (amino acids 232-242) that sticks into the dsDNA and effectively melts it. Adding NTPs in solution then initiates transcription from each promoter synchronously upon binding. The enzyme is very processive - RNAP stays bound to the DNA template and does not have to repeatedly dissociate and rebind. 

Similar to other RNAPs, a short transcript is first made, leading to a "scrunching' of the DNA, before elongation. The synthesized RNA escapes after a large conformational change, which also hides the promoter-binding surface.

In multi-subunit RNAPs, during each nucleotide addition cycle, the trigger loop closes over the correctly paired NTP (ensuring base-pair fidelity and catalytic positioning).  Next, the bridge helix changes conformation with a lever/ratchet driving single-base-pair translocation. In T7 RNAP, a functionally equivalent closure occurs over the NTP.  Figure \(\PageIndex{27}\) shows an interactive iCn3D model of the T7 RNA polymerase elongation complex with bound DNA and RNA transcript (1MSW)

Figure \(\PageIndex{27}\): T7 RNA polymerase elongation complex with bound DNA and RNA transcript (1MSW). (Copyright; author via source). Click the image for a popup or use this external link: https://www.ncbi.nlm.nih.gov/Structu...612b7a02ed2ee7

The double-stranded DNA is shown in ribbons.  The complementary synthesized RNA is shown in spacefill. The N-terminal domain (1-324) has elements (C-helix, H region) that refold significantly during the catalytic cycle, hiding the promoter site and forming an RNA exit tunnel.

• The C-helix (~30-62) is shown as a red cylinder and labeled. 
• The H region (helix-turn-helix or HTH motif, ~151–190) is shown in cartoon, with helices shown as red ribbons. 
• The intercalating β-hairpin (~232–247, middle unresolved) inserts between the DNA strands to melt the promoter during initiation 
• The specificity loop (740–769), with side chains shown in sticks, is labeled. It reads the promoter during initiation, then repositions to interact with the 5' end of the nascent RNA during elongation.
• O-helix (~625–643) closes over the incoming NTP upon correct base-pairing, functionally analogous to the trigger loop's fidelity-checking role
• Specificity loop, residues 740–769, reads the promoter during initiation, then repositions to contact the 5' end of the nascent RNA during elongation. 

This single protein performs all the necessary tasks (promoter recognition, melting, synthesis, termination)! Figure \(\PageIndex{28}\) below shows the conformational changes that occur between two forms of the polymerase.  Here are the PDB files and their descriptions:

• 1CEZ — T7 RNAP bound to duplex T7 promoter DNA (pre-initiation complex without RNA). 

• 1MSW — full elongation complex consisting of RNAP and bound 30 base pairs of DNA  (including the transcription bubble) and a 17-nucleotide RNA transcript. 

Figure \(\PageIndex{28}\): Morphing of 1CEZ polymerase to the 1MSW form, which is the more extended structure (PyMOL). 

Interactive model:  

RNA polymerase (RNAP) is a mechanochemical motor, similar to myosin and kinesin, as we discussed at the end of Chapter 5.5.  It converts the chemical energy of nucleotide bond formation into directed mechanical movement along DNA, similar in principle to motors like kinesin, just walking along a nucleic acid track instead of a filament.

The enzyme is very processive, and full transcription leaves the bound DNA molecule structurally intact. A fluorescently labeled version of the dsDNA (310 bp) can be bound and reused repeatedly as the template for RNA transcription.  Diffusion of the labeled dsDNA can be measured in aqueous solution using  Fluorescence Recovery After Photobleaching (FRAP). A laser photobleaches the fluorophores in a small, defined region, and the time it takes for fluorescently labeled molecules to diffuse back into the region to restore fluorescence emission is measured.  These measurements provide a way to determine the fluorophore's diffusion coefficient, in this case for labeled dsDNA. The rate can be compared to the theoretical rate calculated using an equation for rigid-rod diffusion, assuming the DNA behaves as a short, roughly rod-like polymer little affected by the RNAP. Hence, T7 RNAP with labeled dsDNA can be used to compare the enzyme's translational diffusion rate during active transcription versus the random control rate without transcription.  This can be done in several ways.  One is adding chain-terminating 3'-dNTPs to trap complexes at the initiation stage (rather than letting them run off full transcripts).  Another is by removing Mg²⁺ so NTPs bind, but catalysis can't occur.   Values are compared to a control (absence of NTPs).

Figure \(\PageIndex{29}\) below shows a conceptual interactive simulation of the FRAP experiment (Yu, Jo, Kounovsky, de Pablo & Schwartz, J. Am. Chem. Soc. 2009, 131, 5722–5723).  It shows ten solution-phase (i.e., not attached to a surface) DNA–RNAP complexes in a control (green, left panel) that undergo pure Brownian motion and the DNA-RNAP complex during transcription (blue panel, right), which has the same random jitter and, in addition, a randomly directed mechanical "kick" (yellow flash) each time a simulated RNAP completes a nucleotide-addition cycle. A live averaged squared displacement from each particle's starting position (MSD) vs. time is shown in the graph below the simulation.  Slow the speed slider and restart the simulation.  It is best to view the graphs with repeated restarts, but before the complexes are fully distributed throughout the box.   The simulation shows a ~25% gap emerging over time that matches the diffusion coefficients calculated experimentally in this paper by Yu et al, which describe this solution-phase "molecular propulsion".  Propulsion isn't just a byproduct of making RNA but comes from the mechanical cycling of the motor itself 

Here's a summary of what occurs:  NTPs are added to the growing RNA chain, and pyrophosphate (PPi) is released and hydrolyzed. This provides the energy for the enzyme to translocate exactly one base pair forward along the DNA template.  Chemistry (bond formation) alternates with mechanics (movement), one nucleotide and one step at a time.  In solution, T7 RNAP uses the same stroke to push and pull on the DNA template itself, moving it through the surrounding fluid. Instead of the motor moving along a stationary track, the simulation displays a proxy for the motion: the "track" (the DNA) gets jostled by the motor sitting on it.   

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Figure \(\PageIndex{29}\): Conceptual animation of the molecular propulsion experiment.  Each panel shows ten independent, untethered DNA–RNAP complexes (short rods, RNAP shown as a dark dot) diffusing freely in solution, a group view analogous to the bulk FRAP measurement described in the paper, not a single-molecule trace. In the transcription condition, every simulated nucleotide-addition event delivers a brief, randomly directed mechanical impulse to the DNA on top of the same thermal jitter present in the control. Accumulated over time, these impulses raise the ensemble's apparent diffusion coefficient, illustrating the mechanism the authors call "Molecular Propulsion" and the ~25% average increase they measured by FRAP when transcription was active. Diffusion coefficients and "kick magnitudes" are parameters tuned to produce a comparably sized effect; they are not literal diffusion coefficients (μm²/s).  It's best to view just the initial diffusion from the center and reset the simulations multiple times to show the effect.  Simulation made using Claude (Anthropic).

Summary

(Summary written by Claude, Sonnet 4.6, Anthropic)

RNA is structurally similar to DNA but is predominantly single-stranded, contains ribose rather than deoxyribose, and uses uracil in place of thymine. Despite being single-stranded, most RNA molecules form extensive intramolecular base-paired secondary and tertiary structures essential to their function. The cellular RNA landscape is dominated by rRNA (~90% of total RNA mass), which together with ribosomal proteins forms the ribosome—the site of protein synthesis. rRNA itself has catalytic (ribozyme) activity, specifically peptidyl transferase activity that forms peptide bonds during translation. Transfer RNAs (~70–90 nt) carry amino acids to the ribosome, while mRNA carries the protein-coding message from DNA. A rich diversity of noncoding RNAs (ncRNAs) regulate gene expression at multiple levels, including miRNAs (~20–30 nt) that guide RISC complexes to target mRNAs for degradation or translational repression through Argonaute-mediated RNA interference, snoRNAs that guide rRNA processing, snRNAs that catalyze pre-mRNA splicing, and lncRNAs (>200 nt) that serve diverse regulatory roles with highly cell-type-specific expression patterns.

All RNA polymerases (RNAPs) are multisubunit assemblies sharing a conserved catalytic core. The bacterial RNAP core (α₂ββ'ω) has structural homologs in archaeal and all three eukaryotic RNAPs, and the characteristic crab-claw structure with central cleft, downstream clamp and jaw, bridge helix, trigger loop, and two Mg²⁺ ions at the active site is universally conserved. In bacteria, the association of a sigma factor with the core creates the holoenzyme capable of promoter-specific initiation; different sigma factors (σ70 for housekeeping genes, σ38 under stress) direct the holoenzyme to different gene sets by recognizing the −35 and −10 promoter elements. The key initiating event is flipping of the A−11 base into its σ2 recognition pocket, which triggers promoter melting and transcription bubble formation, after which sigma dissociates, and processive elongation begins.

Eukaryotic transcription by Pol II is vastly more complex. The preinitiation complex (PIC) assembles stepwise from more than 100 polypeptides. TFIID—a ~1 MDa complex of TBP and 13 TAF subunits, 9 of which contain histone fold domains—initiates PIC assembly by binding the TATA box; TBP's four phenylalanine residues insert into the DNA minor groove, bending the helix ~80° and initiating strand separation. Sequential recruitment of TFIIA, TFIIB, TFIIF, Pol II, TFIIE, and finally TFIIH builds the closed complex. TFIIH's XPB ATPase/helicase then introduces torsional strain to open ~11–15 bp of DNA around the transcription start site, positioning ssDNA at the Pol II active site for initiation. The Mediator complex bridges distal enhancer elements to the core promoter through chromatin looping, enabling enhancer-driven transcription that often occurs in bursts. After synthesizing ~20–100 nt, Pol II typically pauses near the promoter before escaping into productive elongation.

During elongation, NTP binding induces trigger loop folding for nucleotide addition, followed by pyrophosphate release and RNAP translocation. Backtracking—displacement of the 3′ RNA end from the active site—can occur at pause-prone sequences or after misincorporation; rescue occurs via intrinsic TL-mediated RNA hydrolysis (slow) or via rapid cleavage stimulated by GreA/GreB factors in bacteria. Prokaryotic transcription termination occurs by two mechanisms: intrinsic termination, in which an RNA hairpin destabilizes the rU:dA hybrid and dissociates the TEC; and Rho-dependent termination, in which the hexameric Rho ATPase-helicase binds cytidine-rich Rut sites on nascent RNA, translocates to pause sites, and disassembles the TEC using ATP hydrolysis. In eukaryotes, Pol II termination of mRNA transcripts requires the AAUAAA polyadenylation signal and proceeds through allosteric conformational changes in Pol II and/or torpedo degradation of downstream nascent RNA by XRN2/Rat1p; ncRNA termination in yeast uses the alternative Nrd1/Nab3/Sen1 pathway to recruit the nuclear exosome.

 

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