10.3: Details of Transcription
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Find a well-written summary of transcription in prokaryotes and eukaryotes at an NIH website (Transcription in Prokaryotes and Eukaryotes). Here (and at this link), you will encounter proteins that bind DNA. Some proteins bind DNA to regulate transcription, inducing or silencing transcription of a gene. We will discuss their role in the regulation of gene expression later. Other proteins interact with DNA simply to allow transcription. These include one or more that, along with RNA polymerase itself, that must bind to the gene promoter to initiate transcription. We will look at bacterial transcription first.
A. Transcription in Prokaryotes
In E. coli, a single RNA polymerase transcribes all kinds of RNA, associating with one of several sigma factor proteins (\(\sigma \) -factors) to initiate transcription. It turns out that different promoter sequences and corresponding \(\sigma \) -factors play roles in the transcription of different genes (illustrated below).
In the absence of the \(\sigma \)-factor, the E. coli RNA polymerase can transcribe RNA, but does so at a high rate, and from random sequences in the chromosome. In contrast, when the \(\sigma \)-factor is bound to the RNA polymerase, the complex seems to scan the DNA, recognize and then bind to the promoter sequence of a gene. In this case, the overall transcription rate is slower, but only genes are transcribed, rather than random bits of the bacterial genome! The Pribnow box, named for its discoverer, was the first promoter sequence characterized.
Elongation is the successive addition of nucleotides complementary to their DNA templates, forming phosphodiester linkages. The enzymatic reactions of elongation are similar to the DNA polymerase-catalyzed elongation during replication.
There are two ways that bacterial RNA polymerase ‘knows’ when it has reached the end of a transcription unit. In one case, as the RNA polymerase nears the 3’ end of the nascent transcript, it transcribes a 72 base, C-rich region. At this point, a termination factor called the rho protein binds to the nascent RNA strand. Rho is an ATP-dependent helicase that breaks the H-bonds between the RNA and the template DNA strand, thereby preventing further transcription.
Rho-dependent termination is illustrated below.
In the other mechanism of termination, the polymerase transcribes RNA whose termination signal assumes a secondary hairpin loop structure that causes the dissociation of the RNA polymerase, template DNA and the new RNA transcript. The role of the hairpin loop in rho-independent termination is illustrated below.
B. Transcription in Eukaryotes
Whereas bacteria rely on a single RNA polymerase for their transcription needs, eukaryotes use three different RNA polymerases to synthesize the three major different kinds of RNA, as shown below.
Note that catalysis of the synthesis of most of the RNA in a eukaryotic cell (rRNAs) is by RNA polymerase I. With the help of initiation proteins, each RNA polymerase initiates transcription at a promoter sequence. Once initiated, the RNA polymerases then catalyze the successive formation of phosphodiester bonds to elongate the transcript. Recall that mRNAs are the least abundant in eukaryotes as they are in bacterial cells.
Unfortunately, the details of the termination of transcription in eukaryotes are not as well understood as they are in bacteria. Therefore, we will focus on initiation, and then consider the processing of different eukaryotic RNAs into ready-to-use molecules.
1. Eukaryotic mRNA Transcription
The multiple steps of eukaryotic mRNA transcription are shown on the next page.
Transcription of eukaryotic mRNAs by RNA polymerase II begins with the sequential assembly of a eukaryotic initiation complex at a gene promoter. The typical eukaryotic promoter for a protein-encoding gene contains a TATA box DNA sequence motif as well as additional short upstream sequences. TATA-binding protein (TBP) first binds to the TATA box along with TFIID (transcription initiation factor IID).
This intermediate recruits TFIIA and TFIIB. Next, TFGIIE, TFIIF and TFIIH, several other initiation factors and RNA polymerase II bind to form the transcription initiation complex. Phosphorylation adds several phosphates to the aminoterminus of the RNA polymerase, after which some of the TF’s dissociate from the initiation complex. The remaining RNA polymerase-TF complex can now start making the mRNA.
Unlike prokaryotic RNA polymerase, eukaryotic RNA Polymerase II does not have an inherent helicase activity. For this, eukaryotic gene transcription relies on the multi-subunit TFIIH protein, in which two subunits have helicase activity. Consistent with the closer relationship of archaea to eukaryotes (rather to prokaryotes), archaeal mRNA transcription initiation resembles that of eukaryotes, albeit requiring fewer initiation factors during formation of an initiation complex.
192 Eukaryotic mRNA Transcription
A significant difference between prokaryotic and eukaryotic transcription is that RNA polymerase and other proteins involved at a gene promoter do not see naked DNA. Instead, they must recognize specific DNA sequences through chromatin proteins. On the other hand, all proteins that interact with DNA have in common a need to recognize the DNA sequences to which they must bind…, within the double helix. In other words, they must see the bases within the helix, and not on its uniformly electronegative phosphate backbone surface. To this end, they must penetrate the DNA, usually through the major groove of the double helix. We will see that DNA regulatory proteins face the same problems in achieving specific shape-based interactions!
2. Eukaryotic tRNA and 5SRNA Transcription
Transcription of 5S rRNA and tRNAs by RNA Polymerase III is unusual in that the promoter sequence to which it binds (with the help of initiation factors) is not upstream of the transcribed sequence, but lies within the transcribed sequence. After binding to this internal promoter, the polymerase re-positions itself to transcribe the RNA from the transcription start site so that the final transcript thus contains the promoter sequence!
5S rRNA by RNA polymerase III is shown below.
3. Transcription of the Other Eukaryotic rRNAs
tRNAs are also transcribed by RNA polymerase III in much the same way as the 5S rRNA. The other rRNAs are transcribed by RNA polymerase I, which binds to an upstream promoter along with transcription initiation factors. We know less of the details of this process compared to our understanding of mRNA transcription. We’ll explore what we do know next. As already noted, transcription termination is not as well understood in eukaryotes as in prokaryotes. Coupled termination and polyadenylation steps common to most prokaryotic mRNAs are discussed in more detail below, with a useful summary at the NIH-NCBI website here.