Post-transcriptional regulation by Transposon control

Question 1

give 4 examples of Post-transcriptional Regulation

Answer 1

1. During transcription anti-termination/attenuation e.g. trp biosynthesis operon
2. At initiation of translation Trans-encoded Regulatory small RNAs-micF and Cis-encoded antisense RNA- Tn10 transposition
3. During translation translational coupling e.g. ompR-envZ

Question 2

properties of Antisense RNA

Answer 2

• diverse
• variable in size: 100-3500nt
• Steady state levels range
• transcription occurs from antisense strand

Question 3

what are the affects of asRNA binding?

Answer 3

1. Alter target RNA stability
2. generate a specific processing site
3. Modulate translation
4. terminate Transcription

Question 4

how does tn10 adapt to the environment to change gene expression?

Answer 4

1. detects high levels of transposase in environment
2. promoters overlap so when both are transcribed form complementary H bonds blocking initiator.
3. ribosome can’t bind.

Question 5

whats an Insertion Sequences?

Answer 5

• 0.75 - 1.5 kb of Inverted terminal repeats
• One ORF
• Multiple types and copies per genome

Question 6

General properties of transposable elements in bacteria

Answer 6

1. Transpose to one or many target sites

2. Transposition frequency low.

Question 7

Beneficial effects of Transposable elements in bacteria

Answer 7

1. Gene activation
2. Gene mobility
3. Advantageous mutations

Question 8

Adverse effects of Transposable elements in bacteria

Answer 8

• Metabolic load
• Insertional in/activation 
• Polarity effects
• Adjacent rearrangements
– deletion
– inversion
– co-integration or replicon fusion

Question 9

what is Tn10?

Answer 9

classic composite Transposon

Question 10

how does Tn10 work?

Answer 10

• Tcr is the tetracycline-resistance gene.
• IS10-L and IS10-R are 1.3-kb inverted repeats of
insertion sequence 10.
• O and I indicate the outside and inside ends.
• pIN is the promoter of the transposase gene (arrow).
• The transposase gene in IS10-L is non-functional.

Question 11

describe RNA-RNA INTERACTIONS

Answer 11

• pIN transcript controlled by RNAout expressed from pOUT.
• 69 bp transcript (usually)
– stronger promoter • 100x
– RNAout&raquo_space; RNAin
– RNAout more stable
• 1⁄2 life: RNAout 70 min & RNAin 0.5 min

Question 12

describe Repression of transposase translation

Answer 12

• RNAout overlaps RNAin complementary to 5’ of RNAin 35 bp overlap in untranslated region
• Interaction of RNAout with RNAin occludes RNAin abs Not complemented in trans
  – Transposase is cis- acting
  RNA out trans-acting repressor of untranslated RNAin

Question 13

RNA-RNA INTERACTIONS IN Tn10 TRANSPOSITION REGULATION

Answer 13

• Repression low when [RNAout] is low
– naïve cell
• Mechanism
– kissing reaction
1. Stem-loopformsinRNAout
2. RNAout loop interacts with RNAin.
3. Stemunannealtoform complete hybrid
• RNAin rbs in dsRNA

Question 14

External Promoters are?

Answer 14

• What if [RNAin] increased relative to RNAout by strong external promoter?
• Affect RNAout repression
• Evolved mechanism to repress rbs use when longer 5’ untranslated region present
• External promoter leads to longer untranslated region
• sequence upstream of pIN encode other part of inverted repeat