10.2: Overview of Transcription

Transcription, the synthesis of RNA, is the first step of the “central dogma” of DNA-to-protein information transfer. As we will see, some RNAs are translated into polypeptides, while others serve functional and even enzymatic roles in the cell. We begin with a look at the main kinds of RNA in cells.

10.2.1. The Major Types of Cellular RNA

All cells make three main kinds of RNA: ribosomal RNA (rRNA), transfer RNA (tRNA) and messenger RNA (mRNA). rRNA is a structural as well as enzymatic component of ribosomes, the protein-synthesizing machine in the cell. So it’s not surprising that rRNAs are by far the most abundant RNAs in the cell. In contrast, mRNAs are the least abundant. Three rRNAs and about fifty ribosomal proteins make up the two subunits of a bacterial ribosome, as illustrated in Figure 10.1 below.

Figure 10.1: Ribosomal RNAs (the 23S, 16S and 5S rRNAs) and as many as 50 proteins make up the bacterial ribosome shown here. This structure probably does not exist in isolation, but only when the subunits unite with an mRNA during translation. Eukaryotic ribosomal subunits are similar, but larger.

tRNAs decode base sequences of mRNAs into amino acids during protein synthesis (translation), thereby converting nucleic-acid-sequence information into the amino-acid sequences of polypeptides. The tRNAs that are attached to amino acids bind to ribosomes do so based on codon-anticodon recognition (Figure 10.2).

Figure 10.2: Complete ribosomes form when the subunits associate with mRNA. Transfer RNAs (tRNAs) that decode the mRNA then 'enter' the ribosome. They are held in place by codon-anticodon complementarity, as well as by tRNA-ribosomal forces.

186 Transcription Overview: Ribosomes and Ribosomal RNAs

187 Transcription Overview: Demonstrating the Major RNAs

In 2009, Venkatraman Ramakrishnan, Thomas A. Steitz, and Ada Yonath received the Nobel Prize in Chemistry for their studies on the structure and molecular biology of the ribosome.

CHALLENGE

Even though bacteria lack nuclei, the existence of mRNAs in prokaryotes was simply assumed. Why?

The fact that genes reside inside the eukaryotic nucleus but that the synthesis of polypeptides (encoded by those genes) happens in the cytoplasm led to the proposal that there must be an mRNA. Sydney Brenner eventually confirmed the existence of mRNAs. Check out his classic experiment in Brenner S. (1961; An unstable intermediate carrying information from genes to ribosomes for protein synthesis. Nature 190:576-581).

Before we look at details of transcription, recall for future reference that multiple ribosomes can load an mRNA and move along it as polyribosomes (or polysomes), translating multiple copies of the same polypeptide (Figure 10.3).

Figure 10.3: Polysomes form when more than multiple ribosomes assemble on an mRNA, initiating protein synthesis, one after the other, and sequentially translating multiple polypeptides.

CHALLENGE

mRNAs are a small proportion of a cell’s RNAs, and they are also unstable compared to rRNAs and tRNAs. Why should this be so?

10.2.2. Key Steps of Transcription

As in replication, in transcription an RNA polymerase uses the template DNA strand of a gene to catalyze synthesis of a complementary, antiparallel RNA strand. Also like replication, transcription is also error prone (though more so!). Here are some differences:

• RNA polymerases hydrolyze ribose nucleotide triphosphate (NTP) precursors, as they link the resulting nucleotide monophosphates (NMPs) to form RNA chains, while DNA polymerases use deoxyribose nucleotide triphosphate (dNTP) precursors.
• RNAs incorporate uridines (the uracil nucleotides) opposite a template adenine instead of thymidines (the thymine nucleotide). Thymidines end up opposite adenines in new DNA.
• In contrast to replication, RNA synthesis does not require a primer. With the help of transcription initiation factors, RNA polymerase locates the transcription start site of a gene and begins synthesis of a new RNA strand from scratch.

CHALLENGE

Despite the many more mistakes made during transcription than replication, why are transcription errors less consequential?

Several of the DNA sequences that characterize a gene are seen below in the summary of the basic steps of transcription in Figure 10.4. The promoter is the binding site for RNA polymerase. It usually lies upstream of (5’ to) the transcription start site (the bent arrow). Binding of the RNA polymerase positions the enzyme near the transcription start site, where it will start unwinding the double helix and begin synthesizing new RNA.

Figure 10.4: The basic steps of transcription are (1) Initiation, the recognition of a promoter sequence near the transcription start site (the bent arrow) by RNA polymerase; (2) Elongation, the successive addition of nucleotides to a growing RNA strand; (3) Termination, the mechanism by which at the end of the gene, the newly made RNA is released.

In each of the three panels, the transcription unit is the DNA region to be transcribed, extending from the start site (the bent arrow to the right of the promoter) to a point just short of the termination site. Termination sites are typically downstream of (5’ to) the transcription unit. By convention, upstream and downstream positions designate 5’ and 3’ regions of a given reference point on the DNA.

188-2 Transcription Overview: The Basics of RNA Synthesis

Some bacterial transcription units encode more than one mRNA. Bacterial operons are an example of this phenomenon. The resulting mRNAs can be translated into multiple polypeptides at the same time. In Figure 10.5 (below) RNA polymerase is transcribing an operon into a single mRNA molecule encoding three separate polypeptides.

Figure 10.5: An operon is a contiguous group of two or more genes in a bacterial genome that are transcribed as a single messenger RNA encoding all of the genes, all of which are then translated into polypeptides.

CHALLENGE

Why do you think some genes are organized into operons in bacterial cells, and why might eukaryotes lack (or have lost) this kind of gene organization?

Transcription of all bacterial RNAs requires only one RNA polymerase. Different RNA polymerases catalyze rRNA, mRNA, and tRNA transcription in eukaryotes. We already noted that Roger Kornberg received the Nobel Prize in Medicine in 2006 for his discovery of the role of RNA polymerase II and other proteins involved in eukaryotic mRNA transcription.

189 RNA Polymerase in Prokaryotes and Eukaryotes

While mRNAs, rRNAs, and tRNAs are most of what cells transcribe. A growing number of other RNAs (e.g., siRNAs, miRNAs, and lncRNAs) are also transcribed. Some functions of these transcripts (including control of gene expression or other transcript use) are discussed in an upcoming chapter.

10.2.3 RNAs Are Extensively Processed After Transcription in Eukaryotes

Eukaryotic RNAs are processed (i.e., trimmed and chemically modified) from large precursor RNAs into mature, functional RNAs. These precursor RNAs (pre-RNAs, or primary transcripts) contain in their sequences the information necessary for their function in the cell.

Figure 10.6 provides an overview of the transcription and processing of the three major types of transcripts in eukaryotes.

Figure 10.6: Eukaryotic mRNAs, rRNAs, and tRNAs are transcribed by different RNA polymerases and are processed (hydrolyzed) by different mechanisms to yield functional RNAs. Pre mRNAs are spliced to remove intron, ‘capped at their 5’ ends and polyadenylated at their 3’ ends (left). Pre-rRNAs are methylated and cleaved based on methylation patterns (middle). Pre-tRNAs processed by trimming their 5’ and 3’ ends and chemical modification of some bases (right).

To summarize the illustration:

• Many eukaryotic genes are “split” into coding regions (exons) and noncoding intervening regions (introns). Transcription of split genes generates a primary mRNA (pre-mRNA) transcript. Pre-mRNA transcripts are spliced to remove the introns from the exons; exons are then ligated into a continuous mRNA. In some cases, the same pre-mRNA is spliced into alternate mRNAs that encode related but not identical polypeptides!
• Pre-rRNA is cleaved and/or trimmed (not spliced!) to make shorter mature rRNAs.
• Pre-tRNAs are trimmed, and some bases within the transcript are modified. Then three bases (not encoded by the tRNA gene) are enzymatically added to the 3’ end.

190 Posttranscriptional Processing; an Overview

The details of transcription and processing differ substantially in prokaryotes and eukaryotes. Let’s focus first on details of transcription itself and then RNA processing.