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Gene transcription and RNA modification - Transcription in bacteria

30 important questions on Gene transcription and RNA modification - Transcription in bacteria

Which study first indicated that DNA was used as a template to synthesize RNA?

Research by Volkin and Astrachan in 1956, they observed that the RNA immediately made after infection of E. Coli cells by T2 bacteriophages had a base composition substantially different from the base composition of RNA made prior to infection. The base composition after infection was also very similar to the base composition in the T2 DNA (except for T/U).

Who proposed the idea that RNA was used as a genetic messenger from DNA to ribosome to provide the information for protein synthesis?

Meselson and Jacob

Who confirmed the hypothesis of Meselson and Jacob about transcription and translation?

Brenner, in collaboration with Jacob and Meselson (1961)

How are the bases in a promoter sequence numbered?

In relation to the transcriptional start site.

How are the bases preceding the transcriptional start site numbered?

In a negative direction, so that none of the bases is numbered zero.

There are two sequences in many promoters which are particularly important for promoter recognition in bacteria. At which numbers in the promoter are these sequences located?

At approximately -35 and -10.

What is the Pribnow box?

The 5'-TATAAT-3' sequence at approximately site -10 in bacterial promoters.

What is the consensus sequence? And what does this practically mean?

The most commonly occurring bases within a specific type of sequence. For example, when the sequences at the -35 and -10 sites in bacteria are more like the consensus sequence for this part of the DNA, the rate of transcription is higher.

Of which parts is the RNA polymerase holoenzyme built?

The core enzyme of RNA polymerase and a sixth subunit, the σ factor.

What is the function of the 2 α subunits within the holoenzyme of RNA polymerase in bacteria?

- The proper assembly of the holoenzyme
- The process of binding to DNA

What is the function of the β and β' subunits?

- Binding to the DNA (same as α-units)
- To carry out the catalytic synthesis of RNA

What is the function of the ω subunit of the holoenzyme of RNA polymerase in bacteria?

The proper assembly of the core enzyme.

What is the primary role of σ factor within the holoenzyme of RNA polymerase in bacteria?

The recognizing of the promoter.

Seen the fact that the σ factor has a role in recognizing the promoter, it influences the function of RNA polymerase. How are proteins that influence the function of RNA polymerase called?

Transcription factors.

How is a promoter identified by the RNA polymerase holoenzyme?

- The holoenzyme encounters a promoter;
- σ factor recognizes both the -35 and -10 sequences;
- The helix-turn-helix motif within the σ factor can bind to these sequences;
- Two α helices of the σ factor fit into the major groove of the DNA double helix and form hydrogen bonds with the bases;
- σ factor within the holoenzyme forms a closed complex when binding to the promoter

For transcription to begin, the DNA must be unwound. Where does this unwinding begin?

At the TATAAT sequence in the -10 site.

Why is the DNA in the TATAAT region more easily separated than other parts of the DNA?

The TATAAT region only has A-T base pairs, and A-T base pairs only form 2 hydrogen bonds and thus are more easily separated.

After a short RNA is made within the open complex, what happens?

σ factor is released and the core enzyme of the RNA polymerase starts with the elongation.

What is an open complex?

The place during the elongation (and the last part of initiation) where the DNA is unwound (approximately 17 bp long)

Which strand is used as a template strand is different vary among different genes. What, however, is always the same concerning the used strands?

The template strand is read in the 3' to 5' direction, and the synthesis of RNA occurs in a 5' to 3' direction.

Which bonding is forced to separate when termination occurs?

The hydrogen bonding between the DNA and RNA within the open complex.

Which two things are released when the RNA-DNA hydrogen bonding is separated in the process of termination?

RNA polymerase and the newly made RNA transcript.

Which two different mechanisms for termination have been identified in E. Coli?

- For certain genes, an RNA-binding protein known als rho protein is responsible for terminating transcripten (p-dependent termination);
- For other genes, p protein involvement is not required (p-independent termination)

What is the rut site?

Rho utilization site, a site in the DNA which enocdes a sequences in the RNA that acts as a recognition binding site for the binding of p protein.

How does p protein function?

As helicase (an enzyme that can separate RNA-DNA hybrid regions). After the rut site is synthesized in the RNA, p protein binds to the RNA and moves in the direction of RNA polymerase.

What is the second component of p-dependent termination?

The site where the actual termination takes place (the termination site).

What happens at the termination site in p-depedent termination?

- The DNA encodes an RNA sequence containing GC-bases that form a stem-loop structure;
- This stem-loop structure binds to RNA-polymerase, which causes RNA polymerase to pause;
- In this pause the p protein catches up to the stem-loop, pass through it and break the hydrogen bonds between the DNA and RNA;
- The RNA strand is then separated from the DNA and RNA polymerase.

The process of p-dependent termination does not require p protein. What does it require then?

Two adjecent nucleotide sequences, the one forms a stem-loop and the other, downstream from the stem-loop, is a uracil-rich sequence at the 3' end.

How does the process of p-independent termination work?

- The stem-loop causes the RNA polymerase to pause;
- Other proteins, like NusA, stabilize the pause by binding to RNA polymerase;
- At that time, the uracil-rich sequence in the RNA is bound to the DNA template strand. The binding of this uracil-rich sequence to the DNA template is relatively weak, causing the RNA transcript to dissociate from the DNA;
- Transcription is stopped.

How is p-independent termination also called?

Intrinsic termination (because no protein has to physically remove the RNA from the DNA).

The question on the page originate from the summary of the following study material:

Genetics: Analysis And Principles | 9781260571226 | Robert Brooker

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