Lecture 6 - What happens after transcription initiation?

Question 1

What are the features of Rho-independent termination of transcription?

Answer 1

• RNA forms stem-loop with Watson-Crick base pairing followed by a string of 5 U’s
• This structure causes RNA polymerase to dissociate from the DNA (by recognising the RNA pol and sticking in it) immediately AFTER transcribing this terminator sequence
• so that transcription elongation is abrogated

Question 2

What are the three stages in translation?

Answer 2

Initiation
-ribosome with aminoacyl-tRNA binds to the Shine-delgano sequence 10 bp upstream of the AUG start codon on mRNA (ribosome binding site)
Elongation
-ribosome moves along the mRNA extending the peptide with one aminoacid per codon
-tRNA is loaded with a specific aminoacyl defined by the anti-codon sequence
Termination
-occurs at one of three stop codons

Question 3

What are unique aspects of prokaryotes?

Answer 3

• no membrane bound organelles (nucleus, golgi, ER or mitochondria)
• primary transcript involves no processing
• transcription and translation are coupled

Question 4

When does translation occur in bacteria in relation to transcription?

Answer 4

Bacterial transcription and translation occur simultaneously
As RNA polymerase transcribes mRNA, ribosome binds and begins to translate the mRNA into protein

Question 5

After initation of transcription how is expression regulated? (At 3 points)

Answer 5

Elongation of transcription
-translation, RNA binding regulatory protein, metabolite (riboswitch)
Translation initiation
-RNA binding regulatory protein, sRNA, metabolite (riboswitch), mRNA stability
Controlling protein activity
-protein folding, interaction with ligand, modification

Question 6

What structures control attenuation and how?

Answer 6

alternative RNA secondary structures

• termination of transcription at intrinsic terminators can be attenuated by controlling the formation of the termination hairpin structure in RNA
• factors (ribosomes, RNA binding regulatory proteins, metabolites) either stabilise or destabilise the terminator sequence

Question 7

What are the features of attenuation of the trp operon?

Answer 7

• Trp is an aa, energy costly to make (expression of the trp operon is required for biosynthesis of Trp)
• expression of trp should be shut off with excess trp and turned on in the absence of trp, therefore expression is tightly regulated
• attenuation controlled by ribosomes

Question 8

What are the features of E.coli trp attenuation?

Answer 8

• controlled by translation (ribosomes)
• attenuator located between the promoter and the first gene of the trp gene cluster
• a leader peptide contains a little ORF, two trp codons and a potential terminator
• the structure of the RNA at the attenuator depends on whether the leader peptide ORF is translated

Question 9

What is the purpose of the leader peptide in E.coli trp attenuation?

Answer 9

• helps the ribosome whilst translating the leader peptide to understand the levels of trp
• no trp the Ribosome pauses which affects downstream translation (leader peptide not translated, terminator sequence does not form)
• lots of trp the R carries on translating the leader peptide and the RNA pol falls off (terminator hairpin forms)

Question 10

What in the leader region of the E.coli Trp gene helps to control termination?

Answer 10

Potential secondary structures:
-leader region has lots of potential secondary structures and alternative secondary structures that can form
There are 4 regions that can form secondary structures:
-Region 2 is complimentary to regions 1 and 3
-Region 3 is complimetary to 2 and 4
-2:3 is an antiterminator - when regions 2 and 3 pair, the terminator region is single stranded
-3:4 pairing forms the terminator region

Question 11

How is attenuation of the trp gene controlled by translation in E.coli?

Answer 11

Through ribosome movement
Absence of trp
-the ribosome stalls at the trp codons (as it has not got a loaded tRNA(trp))
-an alternative secondary structure (2:3) prevents the formation of the hairpin so transcription continues
Presence of trp
-the leader is translated (have loaded tRNA(trp))
-the attenuator is able to form the hairpin that causes transcriptional termination (3:4) and ribosome falls off

Question 12

What function of bacterial translation/transcription make it possible to have attenuation controlled by translation (trp in E.coli)?

Answer 12

coupled translation/transcription

Question 13

Aside from attenuation by translation, what other sensing mechanism is present in E.coli for the trp operon?

Answer 13

repression of transcription initiation by TrpR repressor (trp is the corepressor)

Question 14

What is trp attenuation controlled by in Bacillus?

Answer 14

RNA binding protein

• leader region (like E.coli) at the 5’ untranslated region of transcript has a binding site for the RNA binding protein TRAP
• TRAP= Trp RNA-binding attenuation protein
• TRAP needs to be in a complex with tryptophan to bind
• TRAP controls formation of the terminator in the leader, without Trp an anti-terminator forms

Question 15

What occurs in attenuation of Trp when Trp is low in Bascillus?

Answer 15

Low Trp

• TRAP (RNA binding protein) is inactive because there is insuffient Trp
• Anti-terminator forms during transcription, (a 4/5 nucleotide stem) and transcription continues into Trp genes

Question 16

What occurs in attenuation of Trp when Trp is high in Bascillus?

Answer 16

High Trp

• Trp activates TRAP by binding
• TRAP binds mRNA repeats (regular spaced triplets which are the contact points for TRAP) at the leader region of the trp operon and prevents the anti-terminator from forming
• Terminator forms and transcription terminates

Question 17

Compare the regulation of expression of Trp Operons in E.coli and B subtilis

Answer 17

E.coli
-Initiation: Trp (corepressor) allows repression by TrpR (aporepressor)
-Attenuation: Information on Trp is transmitted by tRNA[TRP] about the Trp concentration; effected through translation/movement of ribosomes
B. subtilis
-Attenuation: Trp activates RNA binding protein TRAP, stops anti terminator forming, terminator forms
-other additional mechanisms

Question 18

What are sRNAs?

Answer 18

Small RNAs (50-500nt in length) that pair with a target transcript forming a duplex region and control expression (increased OR decreased depending on the specific interaction) by:
-affecting translation initation (block access to RBS)
or
-destabilising target mRNA
sRNAs work in trans and the pairing does not need to be perfect
-about 150 in E.coli e.g. OxyS RNA, oxidative stress causes binding

Question 19

Why is it advantageous the sRNAs can work in trans?

Answer 19

In trans: can be encoded on a separate transcript to target

-can be made in different gene/openon than the one being targeted

Question 20

How can sRNAs increased AND/OR decrease expression?

Answer 20

This depends on the specific interaction

Translation repression:
-mostly occludes the RBS -> inhibition of translation (OxysRNA)
-mRNA degradation: binding of sRNA forms a stem and loop structure within the mRNA which is recognised by enzymes that cleave RNA
Translation activation:
-mRNA may form complimentary loop structure through complimentary binding blocking the RBS -> binding of sRNA to a section in this structure prevents it binding to the RBS and allows translation

Question 21

Define attenuation

Answer 21

A mechanism of control for some bacterial operons which results in premature transcription termination

• based on the cotranslation of bacteria
• involves a stop signal (attenuator) located in the DNA segment that corresponds to the leader sequence of mRNA
• ribosome becomes stalled in the attenuator region in the mRNA and then either reads through or stops transcription depending on the metabolic conditions

Question 22

Define anti terminator

Answer 22

A secondary structure that forms in mRNA during transcription and its formation prevents the formation of the terminator, so that ribosome can continue translating the mRNA into protein

Question 23

What are the steps in transcription?

Answer 23

Initiation
-RNA pol finds, binds to promoter and makes an open complex
Elongation
-RNA transcript is extended by the addition of nucleotides
Termination
Transcription ends at a terminator (Rho-dependent and Rho independent terminators)