Lecture 10 Prepared by Belay 08 March 2019

REGULATION OF GENE EXPRESSION IN PROKARYOTES:

The tryptophan operon as an example of gene expression control at the level of RNA elongation

Operon
- is a set of genes that are transcribed under control of a single promoter — regulated and expressed together as
a unit
- Usually contain clusters of genes which code for proteins (enzymes) that are all involved in one metabolic
process
- Operon = promoter + operator + gene cluster
- Usually regulated by repressors and/or activators in response to the environment
- Operons are turned “on” or “off” by a DNA switch — operator
- If operator is open — RNA polymerase can bind
- If operator is blocked — RNA polymerase cannot bind

Tryptophan and bacteria

- bacteria need amino acids to build proteins — one of which is tryptophan
- If tryptophan is available in the environment, bacteria such as E. Coli will take it up and use It to build proteins
- E.coli can synthesise their own tryptophan using enzymes that are encoded by trp operon

trp Operon
- A group of genes in bacteria that encode biosynthetic enzymes for the amino acid tryptophan
- Encodes 5 structural genes that synthesise tryptophan
- Promotes the production of tryptophan when tryptophan isn’t present in the environment
- Tryptophan levels
- Low — trp operon is expressed (turned “on")
- High — trp operon is repressed (turned “off”)
- Regulation
- Operates by a negative repressible feedback mechanism
- trp repressor
- Attenuation

trp Operon genes

trp operon gene Gene function

trp p,O Promoter; operator sequence is found in

the promoter

trpL Leader sequence; attenuator (A)

sequence is found in the leader

trp E Gene for anthranilate synthetase subunit

trp D Gene for anthranilate synthetase subunit

trp C Gene for glycerolphosphate synthetase

trp B Gene for tryptophan synthetase subunit

trp A Gene for tryptophan synthetase subunit

Lecture 10 Prepared by Belay 08 March 2019

Regulation of the trp operon

- expression of the trp operon is regulated at the level of transcription initiation and by attenuation
- Operates by a negative repressible feedback mechanism
- Attenuation — expression of these genes is lessened as a result of this process

• trp repressor
Mechanism
- One tryptophan (corepressor) binds to a site on each monomer of the trp repressor — conformational change
- The trp repressor, a homodimer of two of these complexes, binds to the operator of the Trp operon
- This shuts down the transcription of the 5 genes of the operon so the enzymes used in Trp synthesis are not
synthesized.
- When tryptophan is not present in the cell, the repressor leaves the operator, and transcription of the 5

• Attenuation
- second mechanism of negative feedback in the trp operon — occurs at moderately low levels of trp
- Translation of a leader peptide affects transcription of a downstream structural gene
- Concept to recall: transcription-translation coupling
Lecture 10 Prepared by Belay 08 March 2019

Leader regions of the trp operon (trpL)

- exists upstream of the structural genes
- Function in attenuation of operon expression — attenuator (A) sequence found in the leader
- Attenuator in trp operon contains DNA sequence that causes premature termination of transcription
- Intrinsic terminator
- made up of sequences 3 and 4 — which are able to base-pair to form a G-C rich stem and loop structure
- Other components:
- translation start codon
- Two tryptophan codons (UGGUGG)
- Stop codon found between
- String of U residues at the end of the sequence

Model of intrinsic termination

- Function with RNA pol alone without help from other proteins
- Intrinsic sequences of the transcripts synthesized adopt secondary structures which slows down the RNA pol
— eventually stopping it
- Inverted repeat leads transcript to form hairpin structure
- T-rich region in nontemplate strand produces string of weak rU-dA base pairs holding transcript to
template

Mechanism
1. Excess Tryptophan
- rapid translation of the early trp leader mRNA enabling domain 2 to pair with domain 1 — pause loop
- Rapid initial translation is able to occur with excess tryptophan present because there is a sufficient
quantity of Trp-tRNA available to translate the two Trp codons
- Ribosome pauses at a stop codon, blocking sequence 2, allowing sequence 3 to pair with sequence 4 —
terminator loop
- The terminator loop binds RNA polymerase — releasing it before reaching trpE
- Transcription of the remainder of the trp operon is terminated
Lecture 10 Prepared by Belay 08 March 2019

2. Low Levels of Tryptophan

- slow translation of the early trp leader mRNA — enable domain 2 to pair with domain 3 — anti-terminator loop
- Slow initial translation is able to occur with low levels of tryptophan present because there is limited Trp-
tRNA available to translate the two Trp codons — causing the ribosome to stall at the Trp codons
- Formation of the paired structure between sequences 2 and 3 prevents the formation of the attenuator
structure with sequence 4
- Transcription of the remainder of the trp operon continues and the enzymes required for tryptophan synthesis
are made

Other operons similarly regulated

lac Operon and regulation
-  encodes for the three enzymes needed for the degradation of lactose (for utilization)
- an inducible system
- binding of an inducer alters the shape of the regulatory protein in a way that now blocks repressor binding to
the operator — thus permitting transcription
- lactose functions as an inducer
- absence of lactose — the active repressor protein binds to the operator and RNA polymerase is unable to
bind to the promoter and transcribe the genes
- lactose is present — a metabolite of lactose (allolactose) binds to the allosteric repressor protein and
causes it to change shape — no longer able to bind to the operator; RNA pol is able to bind
- sllolactose is produced from lactose by β-galactosidase (lactase)