Prokaryotic Transcription II

What are the 3 steps in transcription?

initiation, elongation and termination

initiation

setting up. RNA polymerase holoenzyme locates and binds to promoter DNA to begin the synthesis of RNA.

elongation

actual synthesis of RNA. RNA polymerase moves along the DNA template, unwinding the DNA ahead of it and adding RNA nucleotides one at a time to the 3’-OH end of the growing RNA strand.

As RNA polymerase moves along the DNA template, unwinding the DNA ahead of it and adding RNA nucleotides one at a time to the 3’-OH end of the growing RNA strand. What is happening behind the RNA polymerase?

the DNA template is rewound, displacing the newly made RNA from its template strand

termination

ending transcription. terminator sequences are recognized, the separation of the RNA molecule from the DNA template occurs, and the RNA polymerase dissociates from the DNA.

What are the 4 stages of transcription initiation?

1. promoter recognition

2. unwinding of dsDNA (double-stranded DNA) and creation of a transcription bubble

3. abortive initiation

4. promoter escape

promoter recognition (transcription initiation)

RNA polymerase (R) interacts with promoter DNA (P) to form the closed complex (RP_c)

Unwinding of dsDNA and creation of a transcription bubble (transcription initiation)

the duplex DNA around the transcription start site is unwound to create a transcription bubble about 12-14 bp long and form the open complex (RP_o)

the transcription bubble is very similar to…

DNA replication bubble

The conversion of RP_c to RP_o requires the presence of what?

the sigma factor

Initial specific binding to sigma factors to the promoter triggers conformational changes that result in the separation of the 2 strands of DNA and expose a portion of the template strand.

What is the difference between the open complex (RP_o) vs the closed complex (RP_c)

(RP_c) is an initial state where the enzyme binds to double-stranded promoter DNA, while a (RP_o) is an active state where the DNA strands separate to expose the template strand.

abortive initiation (transcription initiation)

RNA polymerase begins synthesizing RNA complementary to the DNA template by adding several nucleotides while remaining bound to the promoter and without moving along the DNA template

True or False: Short pieces of RNA with a few nucleotides long may be repeatedly synthesized and released, without the polymerase leaving the promoter.

True. This is due in part to interactions between the sigma factor and promoter.

True or False: All initiation events are productive.

False

promoter escape (transcription initiation)

• After several abortive initiation attempts, the polymerase synthesizes an RNA molecule from 9-12 nucleotides in length.

• This allows RNA polymerase to break its interactions with the promoter and begin moving along the DNA, transitioning into the elongation stage.

When does the sigma subunit dissociate from the core enzyme?

shortly after transcription initiation when the growing RNA chain reaches about 9 to 12 nucleotides in length

When does transcription elongation begin?

After the sigma subunit dissociates from the core enzyme

Transcription elongation (2nd stage of transcription)

the core polymerase moves along the template strand, unwinding the DNA ahead if it to maintain a transcription bubble of 12-15 base pairs and synthesizing RNA complementary to the template strand of the DNA

Is RNA polymerase highly or slightly processive?

highly. It adds 100s or 1000s of bases to the growing RNA at about 20-50 nucleotides per second without requiring additional apparatus (molecules or proteins)

RNA polymerase vs DNA polymerase

RNA polymerase:

• does not require additional molecules or proteins

• highly processive

• proofreads on its own

• performs limited form of proofreading

• less accurate (higher error rate, 10^-6)

DNA polymerase

• requires multiple proteins (ex. a sliding clamp and clamp loader is needed to keep DNA polymerase on the strand)

• slightly processive

• needs other molecules to proofread

• way more accurate (lower error rate, 10^-8)

Unlike DNA polymerase, RNA polymerase does not have an elaborate proofreading mechanism. However, RNA polymerase can perform a limited form of proofreading during transcription. How does it do this?

When RNA polymerase incorporates a nucleotide that does not match the DNA template, it backtracks along the DNA and cleaves the last 2 nucleotides to remove the error. RNA polymerase resumes RNA synthesis after it cleaves and removes the wrong nucleotides.

Terminator

the signal to the RNA polymerase to stop transcription and dissociate from the template

True or False: The terminator is part of the end sequence that is transcribed.

True, and the promoter is not.

What are the 2 types of terminators in prokaryotes?

intrinsic terminators and rho-dependent terminators

intrinsic terminators

allow termination by RNA polymerase without the help of any additional factors

rho-dependent terminators

requires the assistance of a protein factor called rho (ρ) to terminate transcription

Intrinsic terminators account for approximately what percent of prokaryotic terminators? What about rho-dependent terminators?

50%. ~20%.

What 2 common features do intrinsic terminators have?

1. they contain inverted repeats

2. a stretch of ~7-9 adenines (A’s)

inverted repeats

Sequences of nucleotides on the same strand that inverted and complementary. This sequence when transcribed into RNA can base-pair with each other to form a hairpin.

What is the stretch of ~7-9 adenines (A’s) adjacent to the inverted repeat in the DNA for?

These are transcribed into a stretch of uracils (U’s) in the RNA (or U-rich tract). This permits the RNA-DNA hybrid in this region to come apart, because the base-pairing between A’s in the DNA templates and U’s in the RNA is relatively weak (2 hydrogen bonds). This allows the transcript to be released from the DNA template and from the RNA polymerase

As RNA polymerase transcribes the terminator region, what happens to the newly synthesized RNA?

It folds into a stem-loop (hairpin) structure. This secondary structure causes the RNA polymerase to pause.

What happens after the transcript is released from the DNA template and from the RNA polymerase?

The template strand bases the flip back and base pair with the non-template strand, rewinding the transcription bubble. (becomes double-stranded DNA again)

protein factor rho (ρ)

a helices consisting of 6 identical monomers arranged in an open circle

Rho-dependent termination requires the formation of what structure and why?

a hairpin structure that causes pausing of the RNA polymerase (RNAP)

rho utilization site (rut)

A region of the transcript where rho binds. It is C-rich and poorly structured RNA sequence.

Rho couples with what mechanic to translocate along the RNA until it catches up with the paused RNAP?

NTP hydrolysis

Rho uses helicase activity to do what?

to break the RNA-DNA helix, ejecting the RNAP and RNA from the template

DNA replication vs RNA transcription

DNA replication

• replication bubble opens at the origin and proceeds bidirectionally

• RNA primer required, because DNA polymerase can only extend an existing strand

• DNA polymerase synthesizes DNA in the 5’ to 3’ direction

RNA transcription

• transcription bubble opens at the promoter and proceeds unidirectionally

• No primer required, because RNA polymerase can initiate synthesis from scratch

• RNA polymerase synthesizes RNA in the 5’ to 3’ direction