Lecture 14: Transcription in Prokaryotes and Processing of rRNA and tRNA

What does the sigma factor recognize?

the dsDNA that defines the start site of transcription

What is the promoter?

the site in DNA where RNA Pol binds; the entire region on the DNA required for appropriately regulated transcription initiation

What binds to the promoter in bacterial transcription?

the sigma subunit

What are the regions associated with sigma subunit binding?

-10 and -35 sites

How many base pairs separate the -35 and -10 sites?

17.5 base pairs

True or False: Between the centers of the -35 and -10 regions, there is 2 complete turns of the helix—making these two regions on the face of the helix and a major groove away.

true

What allows the sigma protein to recognize both the -10 and -35 sites at the same time?

both of the regions are on the face of helix

What strand are the -10 and -35 sites on?

the non-template strand

What type of motif does sigma 70 have?

two helix-turn-helix (one in -10 and one in -35)

Where does the c-terminal domain of the alpha portion of the sigma 70 bind?

the UP element

RNA Pol binding and initiating transcription depends on what?

the promoter strength

What is promoter strength?

how close the promoter matches the consensus sequence

True or False: There can be multiple RNA Pols synthesizing RNA from the same gene at the same time.

true

What is the extended -10 region?

additional consensus sequences

What is the UP element recognized by?

the alpha subunit hanging down from the RNA Polymerase

What makes a really strong promoter?

the UP element

What other factors affect protein binding?

• alpha subunit

• sigma factor

• auxiliary proteins can help RNA Pols to bind

What can auxiliary regulatory proteins bind?

DNA and the alpha subunit on the RNA Pol

What do auxiliary protein help with?

help RNA Pols to bind and interacts with sigma factors

What is important to know about the lac promoter?

• Operator sequence overlaps the -10 and -35 sequence

• Lac I repressor recognizes the operator sequence

• Repressor binding to the operator physically blocks the RNA Pol

  • Negative regulator

• -10 and -35 in a lac promoter is not consensus

  • The sequence is weak, so two things need to happen

    • The repressor needs to be gone

    • The CAP protein (sequence-specific) binds upstream and interacts with RNA Pol to recognize the sequence

What is associated with negative and positive regulation?

repessors and activators, respectively

What happens during transcription initiation in bacteria?

• Promoter sequence is recognized by the sigma and is bound by the holoenzyme

  • Closed complex

• DNA around the promoter is unwound

  • Open complex

• Approximately 10 nucleotides are polymerized

  • Pol can “stutter” giving abortive transcripts

• Then a transition takes place → promoter clearance

What facilitates the melting of the promoter region?

sigma has the ability to “flip out” and bind bases in the -10

What are abortive transcripts associated with?

an apparent compression of the DNA

What occurs during abortive transcripts/scrunching?

• Eventually, the built-up energy allows for “promoter clearance”

• RNA Pol lays down 9-10 nucleotides and then gets stuck and rewinds

• The front of the Pol is moving forward but the back isn’t—creating energy that allows the transition into the elongation phase

What happens during transcription elongation in bacteria?

• RNA Pol undergoes another conformational change as it exits the promoter and enters the “elongation phase”

• Sigma dissociates

• Other proteins load onto the Pol

  • Additional proteins increase processivity (block pausing), increase accuracy, and/or aid in termination (like NusA)

• The RNA is synthesized as the complex proceeds along the DNA

What is the average rage of transcription?

50-100 nucleotides per sec but is interrupted by pauses often

What may elongation interruptions be used for?

regulation

What does the holoenzyme for RNA Pol look like?

a hand

Once the sigma factor leaves, what happens?

the Pol is wrapped around the RNA

What type of function does RNA Pol have?

editing function

How does RNA Pol edit?

by backing up and hyrolyzing the incorrect nucleotides

True or False: RNA Pol has pretty good fidelity but DNA Pol’s is higher.

true

What are the two types of transcription termination?

rho independent and rho dependent

What is rho-independent termination?

a hairpin structure in RNA is recognized by RNA Pol, and transcription stops—releasing the RNA

What is rho?

a protein that can attach to RNA and cause the Pols to stop transcription and dissociate

True or False: About half of the terminators in E. coli are rho dependent.

true

How can rho cause transcription termination?

If ribosomes lag, then Rho binds RNA and causes transcription termination at the next pause site

What type of protein is rho?

a helicase that acts on RNA-DNA hybrid molecules

What is important to know about the intrinsic (rho independent) terminator?

• Structure is more important than sequence (except for the run of U bases)

• Dyad symmetry in the DNA

  • Inverted repeat

    • 5’ section on one strand is the same on the 5’ section of the second strand; same with the 3’ sections

  • Causes the hairpin structure

    • Bottom of hairpin is G-C rich, so it’s very stable

Why does a RNA hairpin followed by a run of U’s cause termination?

• Both elements (G/C rich inverted repeat and run of U’s) are needed and they must be adjacent

• RNA-RNA interaction in hairpin competes with RNA-DNA binding in the active site causes a pause

• Run of U’s gives an unstable RNA-DNA hybrid (A-U is the weakest base pair)

• So, the pause gives the instability built into the region of A-U base pairs the time it needs in order to dissociate

True or False: Transcription and translation is coupled in bactiera.

true

What is chasing the Pol in transcription?

the ribosomes

True or False: The messages are covered with ribosomes, so there’s little RNA exposed.

true

What does rho bind to?

exposed ssRNA

What does Rho’s ATPase and DNA helicase activity allow it to do?

to move along ssRNA in a 5’ to 3’ direction and to separate RNA-DNA duplexes

True or False: mRNA in bacteria can be polycistronic.

true

What does polycistronic mean?

mRNA has multiple reading frames that can give rise to different enzymes (B-galactosidase, permease, transacetylase); one mRNA gives rise to multiple proteins

What is polarity?

• A nonsense (polypeptide chain terminating) mutation in a gene stops translation

• Rho will bind to the “naked” RNA and terminate transcription

• Therefore, if the gene with mutation is in an operon, the gene(s) downstream from it is/are never transcribed

• The mutation is said to be “polar” on the downstream gene(s)

True or False: Translational stop mutations can be polar.

true