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1. Prokaryotic gene regulation
Lecture - 4
Trp Operon, Riboswitches

2. The Trp Operon
• E. coli
• Repression + Attenuation
• First repressible Operon to be discovered
• Tryptophan (essential amino acid for humans!)
• Regulation at steps after transcription initiation
• 5 structural genes (tryptophan synthetase)
• Showed that regulatory proteins are not essential
for repression/regulation of transcription

3. Co-repression
Attenuation

4. Attenuation
• 5’ end of the Trp Operon when studied revealed:
- Most mRNA terminate prematurely before first gene
itself (trpE)
- Premature termination is avoided when Trp is limiting
- Fact that transcription and translation are not isolated
events in prokaryotes
- RNA forms alternative structures through inter
molecular base pairing.

6. The leader sequence
• 161 nt of RNA made before RNA pol finds the first codon of
trpE
• Near the end is a transcription terminator; 3,4 forms the
hairpin loop by the RNA itself. This is called the Attenuator
also.
• 1-2, 2-3 binding are possible but are not termination type
• In case of high level of Trp a 139 nt long mRNA only is seen.
• This is because the Attentuator has stopped further
transcription !!
• Then when Trp is low how is the Attenuator avoided??

7. 3 features of the leader sequence
1. Complementarities lead to hairpin region (1 &2) of leader seq
2. Complementarities lead to hairpin region (2 &3) of leader seq
- Both these prevent the termination hairpin loop formation
(3&4)
3. Leader seq contains an ORF for 14 amino acids and precedes a
strong ribosome binding site

8. The leader peptide
• The leader peptide has striking features
• 2 trp codons in a row
• Similarly for other operons

9. Process of attenuation and its avoidance
• Codons for the same amino acid is meant to stop the
ribosome trying to translate the mRNA (leader peptide)
• Scenario 1: When Trp is scarce, there is very little Trp charged
tRNA, thus the ribosome stops at Trp codon. This covers
region 1, leaving 2 to pair with 3 and not 4 (termination); ie 3-
4 impossible = transcription of trp operon

10. • Scenario 2: When enough Trp, then high availability of Trp charged tRNA;
ribosome proceeds forward to 2 and blocks it; This enables 3-4 hairpin
(termination); No transcription beyond leader seq.
• Leader peptide is digested by cellular proteases
• Attenuation Shows:
1. Regulation is possible without regulatory protein
2. Multi-level regulation – Repression followed by Attenuation with
intensity of Trp starvation
3. Attenuation is a robust regulatory mechanism in itself (His, Leu operons)

11. Riboswitches
• Another example of regulation without regulatory
proteins
• Riboswitches are regulatory RNA (leader sequence
forming alternate structures) that sense small molecules
(eg. metabolites) and bind them
• Eg. Methionine containing functional genes in B. subtilis
are controlled by a 200 nt leader untranslated RNA that
forms stem-loop structures related to termination. S-
adenosyl methionine binds to the RNA sequences which
modulates (stabilize termination).
• That is they act as Riboswitches !