Introduction to transcription Flashcards
What are the 3 major classes of RNA that participate in protein synthesis
rRNA, tRNA, and mRNA. All of these are synthesized under the direction of a DNA template during a process called transcription.
Describe RNA polymerase
RNA Polymerase (RNAP) is the enzyme that is responsible for the DNA-directed (DNA is the template) synthesis of RNA. The enzyme couples together the ribonucleoside triphosphates- ATP, UTP, CTP, and GTP- on a DNA template in a reaction that is driven by the release and subsequent hydrolysis of pyrophosphate (PPi): (RNA)n residues+ NTP _- (RNA)n+1 residues + PPi. All cells contain RNAP.
Briefly compare RNAP in bacteria and in eukaryotes
In bacteria, one species of RNAP synthesizes all the RNAs in the cell, except for the RNA primer that is needed in lagging strand DNA replication which is synthesized by primase. Eukaryotic cells contain 4 or 5 RNAPs that each synthesizes a different class of RNA. First, we will look at prokaryotic RNAs. Ecoli’s holoenzyme consists of the subunit composition alpha2betabeta’wo-. Once RNA synthesis has been initiated, the o- subunit dissociates from the core enzyme, alpha2betabeta’w which carries out the actual polymerization process.
List the functions of RNAP
Electron micrographs show that RNAP, which has a large size, binds to DNA as a protomer. The large size of RNAP is probably because of the holoenzyme’s several complex functions including template binding, chain initiation, chain elongation, and chain termination.
Explain the region of template binding in E.coli
RNA synthesis is initiated only at specific sites on the DNA template. RNA polymerase binds to its initiation sites through base sequences known as promoters that are recognized by the corresponding o- factor. Nomenclature: base pairs in the promoter are assigned + or - numbers relative to the initiation sites. If the base pairs are upstream (left) of the initiation site, a negative number will be assigned. If the base pairs are downstream(right) of the initiation site, a positive number will be assigned. RNA synthesis occurs in the 5’-3’ direction(the template is 3’-5’). RNAP binds tightly to the promoters and therefore protects the DNA from being degraded by Dnase I. Sequence determinations of the protected regions have revealed the consensus sequence in the promoter. Conserved regions in the promoter include the -10 region called the Pribnow box. Another conserved region was found at -35 and it is most evident in efficient promoters. The sequence between -10 and -35 is not important, but the length is critical- it ranges from 16-19 bp. The initiating (+1) nucleotide, which is nearly always A or G and is contained in a poorly conserved CAT or CGT sequence It was also discovered that some highly expressed genes contain an A and T rich segment between 40 and -60 called the upstream promoter element (UP element). The UP-element containing genes include those that encode rRNAs. The rates at which genes are transcribed are dependent on the rate that the promoters form stable initiation complexes with the holoenzyme.
Explain the process of template binding
The RNAP binds around the -10 and -35 regions which results in ‘melting out’ or separation of the dsDNA of the promoter- this is when a region of about 14bp extending from the middle of the -10 region to just past the initiation site, thereby forming a transcription bubble. The need to form this transcription bubble explains why promoter efficiency decreases with the number of GC base pairs in the -10 region as GC base pairs in this region increase the difficulty to open the helix. So promoter efficiency is highly dependent on GC content.
Reminder: the DNA strand that is the template for transcription is the noncoding or antisense strand(3’-5’). The synthesized strand corresponds with the sense strand- same sequence as transcribed RNA just with Ts instead of Us.
Explain chain initiation
Often after initiating RNA synthesis, the RNAP will only synthesize about 10 bp then release the new RNA- this is called abortive initiation. When RNAP initiates transcription, it keeps its grip on the promoter (on the nontemplate strand). Consequently, conformational tension builds up as the template strand is being pulled through the RNAPs active site, a process called scrunching. We therefore see an increased size of the transcription bubble which must be accommodated within RNAP. In abortive initiation, the RNAP fails to escape the promoter instead it releases the RNA. RNAP then reinitiates transcription from the +1 position. For successful initiation, the promoter must be removed from the RNAP which requires the dissociation of the o- factor.
Describe the structure of E.coli RNAP
The overall shape of RNAP resembles that of a crab claw. The two pincers are formed by the beta and beta’ subunits. In an open conformation, the space between the pincers is occupied by dsDNA. The template dsDNA continues to the active site at the end of the channel. It forms base pairs with the incoming NTP close to the bound Mg+. Then the newly synthesized RNA exit at the RNA exit channel. The o- subunit extends across the top of the core of the holoenzyme. DNA binds to the o- units at the promoter elements (-10 and -35). In the closed conformation (when the pincers are closed), the DNA does not bind to the enzyme
Explain chain elongation
Chain elongation involves the addition of NTP to the 3’-end of the growing chain. This happens in the 5’-3’ direction. During transcription, the dsDNA template has to be opened up to allow for RNA synthesis. Therefore, the RNA chain only forms a short length of RNA-DNA hybrid duplex. Transcription leaves the intact template dsDNA and produces ssRNA. The unpaired transcription bubble of DNA in the open initiation complex travels along the DNA with the RNAP, there are 2 ways this might occur:
> RNAP follows the template strand in its helical path which implies that the RNA wraps around the DNA. This is unlikely since RNA can not unwind.
> RNAP moves along a straight line while DNA rotates. In this model DNA and RNA are not entangled. This promotes supercoiling- the DNAs helical turns are pushed ahead of the advancing transcription bubbles so as to more tightly wind the DNA ahead of the transcription bubble (promotes positive coiling) and the DNA behind the bubble becomes unwound (promotes negative supercoiling).
Explain the statement- transcription occurs rapidly
The in vivo rate of transcription is 20 to 70 nucleotides per second. Once an RNAP has initiated transcription and moved away from the promoter, another RNAP can then bind. The synthesis of RNAs that are needed in large quantities, e.g. rRNAs, is initiated as often as sterically possible. This is accomplished without a clamplike structure like the sliding clamp of DNA pol III. However, the RNAP functions as a sliding clamp by binding to the DNA-RNA complex.
Describe chain termination
Around half of the transcriptional termination sites in E.coli are intrinsic or spontaneous terminators, they induce transcription without assistance. The sequences of these terminators share two common features:
> A region of 7-10 A.Ts with the As on the template strand. Transcription is terminated within or just past this region.
> A G+C-rich segment with a palindromic sequence that is immediately upstream of the A.T region. It forms a hairpin structure.
The stability of the terminators G+C-rich hairpin and the weak base pairing of its oligo(U) tail to template DNA are important factors ensuring proper chain termination. RNAP pauses at the hairpin.
Explain the Rho factor
About half of the termination sites in E.coli are unable to form a strong hairpin structure. For termination these sites require the participation of a protein called the Rho factor. The Rho factor belongs to the same RecA family of hexameric helicases. It enhances the termination efficiency of spontaneously terminating transcripts and induces the termination of nonspontaneously terminating transcripts. In the presence of the Rho factor, termination is as follows: the protein requires a specific recognition sequence on the newly transcribed RNA, upstream of the termination site. Rho factor binds to this recognition sequence called rut- it is a C-rich segment of about 40 nt. It translocates along the ssRNA in the 5’ to 3’ direction until it encounters the RNAP- this reaction is energy-dependent. RNAP is paused at the termination site. The Rho factor unwinds the RNA-DNA hybrid by pushing the RNAP forward. Therefore, it partially rewinds the dsDNA helix at the transcription bubble while unwinding the RNA-DNA hybrid. As a result, the RNA is released.
List the different eukaryotic RNA polymerases
- RNA polymerase I: also called Pol I and RNAP A, is located in the nucleoli and synthesizes precursors of most rRNAs
- RNA polymerase II: also called Pol II and RNAP B, is located in the nucleoplasm and synthesizes mRNA precursors
- RNA polymerase III: also called Pol III and RNAP C, is located in the nucleoplasm and synthesizes the precursors of 5S rRNA, the tRNA, and a variety of other small nuclear and cytosolic RNAs.
Which RNA polymerases have greater complexity- eukaryotic or prokaryotic
Eukaryotic RNAPs have greater subunit complexity than those of prokaryotes. These subunits are also highly conserved across species from yeast to humans-
