Post-transcriptional regulation by Tryptophan biosynthesis and regulatory RNA

Question 1

when does Post-transcriptional Regulation occur?

Answer 1

1. During transcription by attenuation.
2. initiation of translation– Trans-encoded Regulatory small RNAs and Cis-encoded antisense RNA
3. During translation by translational coupling

Question 2

describe Transcriptional Attenuation

Answer 2

1. controls gene expression just after transcriptional initiation as RNA complex progresses through 5-untranslated region (5-UTR)
2. 5`UTR of some genes can form terminator structure before the functional protein coding sequence

Question 3

how does tryptophan Post-transcriptionally regulate?

Answer 3

1. tryptophan operon can be switched off when tryptophan binds to trp repressor protein.
2. tryptophan is a co-reppressor.
3. structural proteins not transcribed.

Question 4

explain transcriptional attenuation in the trp operon

Answer 4

1. the leader sequence contains 4 palindromic sequences.
2. at high [trp] when 1-2 and 3-4 bind theres a intrinsic terminator of transcription that binds RNA pol.
3. low [trp] 2-3 hairpin transcription continues.

Question 5

When levels of tryptophan are low?

Answer 5

• Ribosome encounters codons encoding tryptophan
• low levels of tryptophan means low levels of tryptophanyl-tRNA •Ribosome temporarily stalls
• Ribosome not open 2-3 hairpin loop
• RNAP progresses thru past stem 4 and into structural genes

Question 6

Why multiple regulatory controls?

Answer 6

• Control of tryptophan biosynthesis:
• Tryptophan regulatory molecule – TrpR- initiation control
– Feedback control
• tRNATRP regulatory molecule – Attenuation
• Conditionsoccurwhenseparateeventscould affect levels of tryptophan and tRNATRP separately

Question 7

When levels of tryptophan are high?

Answer 7

• Ribosome encounters codons encoding tryptophan
• high [tryptophan] means high [tryptophanyl-tRNA]
• Ribosome opens 2-3 hairpin loop
• 3-4 hairpin termination loop forms and transcription terminates

Question 8

Other examples of attenuation?

Answer 8

• Nutrient biosynthesis and import operons: – Amino acids, metal ions, nucleic acids
• tRNA synthesase genes-e.g.phe,thr

Question 9

what are the two types of non-coding RNA?

Answer 9

1. cis-encoded antisense RNA

2. Trans-encoded small regulatory RNA

Question 10

what are small RNAs?

Answer 10

• Un-translated RNA molecule 50-200 nucleotides
• encoded by cis ORF
• Expressed under specific environmental conditions

Question 11

explain Positive regulation by regulatory RNA

Answer 11

1. Prevent formation of structure inhibiting translation
2. Ensure processing of 5` end of mRNA
3. Stabilise mRNA by blocking ribonuclease access

Question 12

what is Hfq- RNA binding protein?

Answer 12

an RNA chaperone

Question 13

properties of Hfq protein

Answer 13

• Highly abundant in Gram-negatives
  • Highly conserved in prokaryotes
  • Hfq has high similarity to eukaryotic Sm-like proteins
  • Acts as a hexamer-forms ring-likes tructure with central pore
  • Hfq mutants have pleiotropic effects

Question 14

what does Hfq- RNA binding protein do?

Answer 14

• Stabilises RNAs
• Required for sRNA to interact with target mRNA
• Modulate stability of mRNAs
• Most Hfq regulation is negative as interaction with sRNA results in decreased translation or degradation of mRNA

Question 15

Advantages of RNA regulators in stress situations

Answer 15

• Reduced metabolic costs
• Provides additional levels of control
• Act at post-transcriptional level so ensure fast response
• Unique regulatory properties- coupled degradation of mRNA:sRNA pairs
• Regulate target at different levels-protein and RNA, reduces leakiness and alters dynamics
• Evolutionary advantages –limited base pairing between target and regulatory RNAs allow for flexibility in evolving new targets

Question 16

what does Small RNA-micF do?

Answer 16

1. micF regulates expression of OmpF porin
2. Small RNA thought to provide another point where other regulators can affect porin expression
3. Larger porin size make small molecules that are detrimental to cell more likely to enter
4. Not necessarily change osmolarity detected by EnvZ
5. examples–antibiotics,bilesalts,salicylate, oxygen stress, EtOH, temperature

Question 17

Genetic evidence of post transcriptional control

Answer 17

• increase in micF expression reduces ompF expression
• but deletion of micF does not greatly affect ompF expression
• Whereas deletion of a transcriptional repressor would result in constitutive expression of ompF

Question 18

how is micF regulated?

Answer 18

• micF transcription is regulated in response to environmental signals
• micF promoter has number of binding sites for transcriptional activators
• micFtranscriptioninducedby:
• SoxRS - oxidative stress
• MarA–weakacids
• Rob–cationicpeptideantibiotics
• OmpR–highosmoticstress
• Also induced by temperature and rich nutrient growth conditions

Question 19

give an example of translational coupling

Answer 19

In OmpR-EnvZ system:
– 1000 OmpR molecules/cell
– 10 EnvZ molecules/cell
– co-transcribed from one promoter – one transcript
• poor re-initiation of translation leads to polar effect on expression
• fine control of expression levels but fixed

Question 20

how bacterial gene expression can be controlled following initiation of transcription?

Answer 20

– Attenuation
– Antisense RNA
– Translational coupling

Question 21

two additional mechanisms bacteria can use to respond to environmental change?

Answer 21

Attenuation/Antisense

Question 22

what is micF?

Answer 22

• gene transcribed divergently from ompC
• 93bp RNA
• ~70% complementary to RBS of ompF mRNA
• Model:micF/ompF RNAs form a duplex
• Duplex shields ompF RBS and AUG
• Inhibits translation initiation