Regulation of Gene Expressionin Prokaryotes III

Question 1

The trp operon:

Answer 1

• A repressible system
• E. coli has the ability to both synthesise its own tryptophan and take
  it up (from the environment)

Question 2

Presence of tryptophan in environment results in

Answer 2

coordinated repression of tryptophan synthesis

Question 3

5 contiguous structural genes in the tryptophan operon:

Answer 3

trpE, trpD, trpC, trpB, trpA

• Upstream from these genes, near the operon, are the cis-acting
  regulatory elements
• Includes the:
  > Promotor
  > TrpP
  > Operator region trpO
  > A leader sequence
  > Attenuator sequence
• trP gene encodes repressor protein - located away from the operon
  and chromosome
  = shows that trans elements don’t have to be closely associated
  with the genes they control

Question 4

Functioning of the trp operon
Hypothesis

Answer 4

– inactive repressor cannot interact with operator
= RNA polymerase is free to transcribe the operon into
polycistronic mRNA containing coding sequences for the 5
structural genes

Question 5

Functioning of the trp operon

Answer 5

• Like the Lac repressor - the trp repressor is also an allosteric
  protein
• Binding to tryptophan induces a conformational shift = allows the
  repressor to bind to the operator region
  –> Tryptophan = co-repressor of the system
• Thus, when tryptophan (end point of the biosynthetic pathway) is available in sufficient quantities, the tryptophan repressor complex binds to the operator region and blocks further transcription
• The regulatory region inhibits transcription = negative control
  > Negative feedback loop

Question 6

Negative feedback loop

Answer 6

• End product of the system acts to repress its own synthesis when it is produced in sufficient quantities or when it is readily available in the growth medium.

Question 7

listen to slide 6!

Question 8

Leaky transcription

Answer 8

• In presence of low levels of tryptophan, transcription of the
  upstream region may be initiated = expression is leaky
• Once transcription is initiated, theres an independent
  mechanism to efficiently repress the expression of the operon if
  tryptophan is present = Attenuation
  – transcription terminates after 140 nt at a point just before the
  structural genes
  > this up stream molecule = 5’ leader sequence
  - acts as a Riboswitch

Question 9

watch slide 8

Question 10

Riboswitches

Answer 10

• regulate gene expression
• Structures within mRNA leader transcript that regulate gene
  expression
  > Mostly found in bacteria (5% of Bacillus subtilis genes)
  > One type found in plants and fungi
  > Not found in animals, but existence in animals cant be
  totally ruled out

Question 11

Riboswitches bind to:

Answer 11

• Small molecules
  Vitamins
  Amino acids
  Purine nucleotides (A, G)
  sugars
  Metal ions
  and other small ligands

Question 12

Binding of ligands to riboswitches

Answer 12

= Causes a conformational change
- affects secondary structure of mRNA
- affects ability of RNA polymerase to complete transcription of
that gene

Question 13

most riboswitches (attenuators) are inhibitory

Answer 13

• they are employed by genes for biosynthetic enzymes/
  transporters
• whose expression is inversely proportional to the concentration
  of their corresponding metabolite

Question 14

One of the oldest regulatory systems

Answer 14

= attenuators
- prevalent in many bacteria
- provides fast and sensitive regulation of gene operons
- Commonly used to repress genes in presence of own product or
a downstream metabolite

Question 15

mRNA elements present in the 5’-UTR (untranslated region), upstream of the coding sequence

Answer 15

Riboswitches

Question 16

What is the function of the UTR sites on mRNA

Answer 16

• provides ribosome binding site
• site for the riboswitch

Question 17

Riboswitches contain two important domains

Answer 17

Ligand binding → conformational changes in two RNA domains:
1. Aptamer (binds ligand)
2. Expression platform (forms terminator structure)

Question 18

Depending on the ligand binding status of the riboswitch, the Metabolite-sensing sequence can adopt one of two conformations

Answer 18

1. In the absence of the ligand
   = Anti-terminator (default conformation)
   Terminator

Question 18

Depending on the ligand binding status of the riboswitch, the Metabolite-sensing sequence can adopt one of two conformations

Answer 18

1. In the absence of the ligand
   = Anti-terminator (default conformation)
   - allows RNA polymerase to read through the expression platform
   into the coding region
2. When the ligand is bound to the aptamer
   = induces Terminatorconformation
   - blocking RNA polym. and terminating transcription before it gets
   to the coding region

Question 19

listen to slide 12 and 13