Lecture 9 - Transcription machinery: RNA polymerase and regulatory sequences

Question 1

What is a transcription unit?

Answer 1

A transcription unit is a sequence of DNA transcribed into a single RNA,
starting at the promoter and ending at the terminator
Transcription unit can comprise one gene or multiple genes (because we have polycistronic operons in bacteria)

Question 2

Describe bacterial RNA polymerase

Answer 2

Bacterial RNA polymerase is a multi-subunit enzyme.
It contains a channel or groove that could be the region where DNA is
accommodated
The length of the groove can hold 16 bp
The groove is lined with positive charges
Positively charged amino acids lining the groove have electrostatic interactions with negatively charged phosphate groups of DNA.
Exists in two forms: core and holoenzyme
Holoenzyme has all core subunits plus sigma subunit - “full” enzyme”. Sigma factor enables RNA polymerase to recognise promoter sites along the DNA. The core enzyme is a protein that has affinity for the DNA so is able to bind DNA and find a stable complex with the DNA - can persist for over 60 minutes. However, it is not able to recognise promoter sites - this property is conferred to RNA polymerase by the sigma subunit.

Question 3

Describe the stages of the transcription reaction

Answer 3

Initiation - Includes all the steps of transcription up to
synthesis of the first bond in RNA (RNA pol
finding the promoter, binding of RNA pol to the
promoter, melting the DNA to form the
transcription bubble).
Elongation - Is the stage during which the RNA transcript is extended by the addition of ribonucleosides. Elongation rate - 40 nucleotides per second
Termination - Is the step that ends transcription by stopping the addition of ribonucleosides and causes
the dissociation of RNA pol from DNA and the
release of the RNA transcript.

Question 4

Describe the steps of initiation

Answer 4

1. Holoenzyme binds DNA; closed binary complex
2. DNA melting; open binary complex
3. Ternary complex; abortive initiation
4. Promoter clearance;
   start of elongation; sigma may be released

When the DNA is not yet denatured it is called a closed binary complex
Ternary complex is RNA polymerase RNA and DNA
Abortive initiation is the process through which RNA polymerase starts to transcribe the DNA, produces a very short transcript of 8-10 nucleotides and then this transcript is released and RNA polymerase starts transcribing again the same region of DNA. This is common in bacterial transcription - related to strength of the promoter. When the promoter is very strong and there is very high affinity for RNA polymerase the enzyme is stuck on that region of DNA and is reluctant to move forward and transcribe the rest of the DNA. Eventually RNA polymerase extends 1 mRNA transcript and then moves on, clears the promoter and moves on downstream to transcribe the rest of the gene. At this stage there is promoter clearance which is the start of the elongation step - the sigma subunit may be released as its main purpose is recognising promoters

Question 5

What are the functions of the sigma factor?

Answer 5

promoter recognition
promoter melting
decreasing the affinity of RNA pol holoenzyme for random sequences

Question 6

What are the two termination mechanisms?

Answer 6

Rho-dependent:
Rho helicase
Site recognized by Rho
Possible auxiliary sequences

Rho-independent
Terminator sequence
(intrinsic terminator)

Question 7

Describe Rho-independent termination

Answer 7

Intrinsic terminators possess 2 structural features:
two symmetrical GC-rich segments
a downstream run of 4 to 8 A residues
Relies on a terminator sequence in the DNA which is transcribed in the RNA
When the RNA polymerase is transcribing an GC-rich region it tends to pause because more energy is needed to break the 3 bonds between G and C and so this gives time for the transcript that has already been transcribed to form a stem- loop structure in which the symmetrical GC-rich region forms the stem. This stem-loop structure in the ternary complex is awkward because it makes the continuation of transcription difficult. Then when RNA polymerase is transcribing this stretch of A it can fall apart because of the steric hindrance of the stem-loop structure and release the transcript so transcription is terminated.

Question 8

Describe Rho-dependent translation

Answer 8

It requires:
1. a protein called Rho, that is a hexameric helicase;
2. a sequence known as rut site (rho utilization)
(3. possible ‘auxiliary sequences’)
Rho is a hexameric helicase and ATPase - needs 6 subunits of Rho to form a ring
Rut site is a sequence that is recognised on the RNA transcript by the Rho protein. In each subunit of Rho there is a C- terminal domain that has ATPase activity whereas the N-terminal domain is responsible for binding RNA. Before binding RNA the hexameric Rho is a gapped ring but upon binding RNA it becomes a closed ring. It binds RNA all throughout the different subunits, but the very start, the 5’ of the RNA transcript is bound to a site that is internal in the protein.
The Rho binds the rut site in the RNA transcript and using the ATPase activity it translocates/moves on the RNA transcript until it reaches RNA polymerase - takes advantage of RNA polymerase pausing in the process of transcription. When it reaches RNA polymerase it unwinds the RNA-DNA transcript, the RNA is released and RNA polymerase falls apart so the ternary complex is disassembled