Lecture 25: 10/23/06 RNA polymerase and Transcription

• Previously we saw the mechanism of DNA replication by DNA polymerases.

• During replication, DNA polymerases copy DNA genomes, reverse transcriptase or RNA dependent DNA polymerases makes a DNA copy from animal viral RNA genomes, and RNA dependent RNA polymerases make RNA copies from plant RNA viral genomes

• Likewise, RNA synthesis, i.e. transcription by RNA polymerase, is one of the landmark events in every cell..

• The RNA polymerase makes a RNA copy of the DNA.

RNA polymerase:

• In prokaryotes the RNA polymerase is made up of the core polymerase and sigma factor, both combine to form the holoenzyme.

• Eukaryotes use three different types of RNA polymerases to transcribe their coding and noncoding RNA’s.
1. RNA polymerase I
2. RNA polymerase II
3. RNA polymerase III.

These three RNA polymerases perform different tasks.

• All RNA polymerases like DNA polymerases require a primer to start the synthesis of RNA, although in this case the primer is a single nucleotide riboATP.

• ATP is abundant in cells and thus riboATP added to prime the reaction.

• Transcription requires that the RNA polymerase binds to the DNA at specific transcription start sites.

Transcription in prokaryotes

• The sigma factor recognizes and binds to the promoter region or the TATAA box first after a short stretch of the DNA double helix is unwound.

• Promoters are sequences of DNA that direct the RNA polymerase to the proper initiation sites for transcription.

• When the promoter is bound by sigma factor - core polymerase complex transcription is initiated.

• The promoter sequences in prokaryotes are found -10 and -35 sequence upstream of the start site.

• If there is no sigma factor it was shown that the core polymerase binds to any place in the DNA, reiterating the fact that sigma factor dictates the specific place for transcription initiation.

• The sigma factor is also called as transcription factor and there are atleast a dozen transcription factors in bacteria.
• For e.g.: Bacillus subtilis a gram positive bacterium has specific sigma factors that gets activated to transcribe the genes that are responsible for expressing sporulation components.

• The sigma factor is weakly associated with the core polymerase and thus can be separated by chromatography.

• So, once the core polymerase binds to the transcription start site the sigma factor dissociates.

• The RNA polymerase adds the ribonucleotide in order to the first riboATP and forms a bond to the –OH group with the release of PPi.

Transcription in Eukaryotes:

• Transcription in eukaryotes is much more complex and highly regulated than in prokaryotes.

• A main difference between eukaryotes and prokaryotes is that
• The transcription and translation are coupled whereas in eukaryotes they are separate.
• In eukaryotes the mRNA made from the DNA template is transported outside the nucleus with the help of its polyA tail and gets translated into a protein.

• Like prokaryotes, eukaryotes rely on conserved sequences in DNA regulation.

• However, in eukaryotes the promoter elements are not in –10 and –35 upstream of the start site but found at a variety of places in the DNA upstream or downstream of the start site than in prokaryotes.

• The transcription in eukaryotes is carried out by three different RNA polymerases.

• They are RNA polymerase I, II and III.

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  These three polymerases have different template specificity, location in the nucleus and inhibitors.

• RNA polymerase I- makes rRNA from rDNA genes.
• The promoter sequences are located in the stretches of DNA separating the genes.
• It has a TATA like sequence at the transcription start site called the ribosome initiation region.
• Further upstream 150 to 200bp from start site are the upstream promoter element.
• Both UPE and rInr aid in transcription by binding proteins that recruit RNA polymerase I.

• RNA Polymerase II – produces pre-mRNA, also called either heterogeneous nuclear RNA or hn-RNA.
• The promoter sequence are set of conserved sequences at various distances upstream of start site and also has an enhancer element that can be very far (more than Kb) from the start site.

Figure 1 Comparison of a simple eukaryotic promoter and extensively diversified metazoan regulatory modules. a, Simple eukaryotic transcriptional unit. A simple core promoter (TATA), upstream activator sequence (UAS) and silencer element spaced within 100–200 bp of the TATA box that is typically found in unicellular eukaryotes. b, Complex metazoan transcriptional control modules. A complex arrangement of multiple clustered enhancer modules interspersed with silencer and insulator elements which can be located 10–50 kb either upstream or downstream of a composite core promoter containing TATA box (TATA), Initiator sequences (INR), and downstream promoter elements (DPE).

• RNA Polymerase III- makes tRNA and other small RNA’s.
• Unlike in the case of RNA Polymerase II promoters, promoter for RNA Polymerase III and I are within the transcribed sequence downstream of the start site.
• It has two types of intergenic promoters.