Molecular Biology II Transcription Regulation in

Bacteria

The lac Operon:

The first regulatory mechanism found
The lac repressor mechanism controls the production of 3 proteins involved with lactose
metabolism

The lac operon contains 3 genes

 lacZ : β-Galactosidase – intracellular enzyme that cleaves lactose  glucose + galactose
 lacY : Lactose Permease – membrane-bound transport protein that pumps lactose into cell
 lacA : Transacetylase – transfers an acetyl group from acetyl-CoA to β-Galactosides

The
lacI
gene

codes
for the lac
repressor – a
homo-dimer
The lac repressor binds tightly to the operator region within the lac operon, which is found
upstream of the 3 genes involved in lactose metabolism
The repressor binds DNA as a homodimer, at 2 locations (once on each strand)
 There is a palindromic sequence centred at +11
 Each homodimer binds either side of this on a different strand
 The key repressor binding sequence (O1) overlaps with the transcription start site
o So repressor binding blocks transcription – RNAP cant bind to promoter
o RNAP and repressor cannot bind at the same time

If lactose binds to the repressor, it can no longer bind to the operator, which allows RNAP to
transcribe the gene
The repressor will bind to the main operator region O 1 (+11) and to one of two ancillary operators

Lecture 18 [Page 1]
Molecular Biology II Transcription Regulation in
Bacteria

 O2 – Inside the transcribed region. Acts as an additional roadblock to prevent RNAP from
transcribing the lac operon, centred around position +412
 O3 – Upstream. -82

When the 2 dimers in the repressor bind to the

operators, they form a tetramer and an
intervening DNA loop is formed. The hinge
between the 2 dimers is flexible. If the hinge is
open then DNA is unstressed, and in B-form. A
closed hinge results in overwound DNA.

The formation of the loop stabilises the

repressed state of the lac operon

A loop between O1 and O3 will make the

promoter inaccessible to RNAP

A loop between O1 and O2 will stabilise the ‘roadblock’

All 3 operators are required for effective repression. This was studied by removing an operator in
an organism then studying the levels of repression. Removing O 1 has the most substantial effect –
a 1000 fold decrease in repression.

Transcription Termination:

Important to terminate
 Reduces energy waste
 Prevents read-through into adjacent genes
 Prevents RNAPs crashing into one another (not all genes are transcribed in the same
)

I) Unconditional Termination – ρ (rho) independent

~50% mRNA termination for protein encoding genes and most tRNA / rRNA transcripts
Carried out using RNAP only, no additional factors
Depends on the formation of a stable, G-C rich hairpin structure in the emerging transcript,
followed by a uracil rich region.
This structure results in a destabilised elongation complex, and disruption of essential protein-
nucleic acid interactions between RNAP / RNA / DNA (U-A base pairs are weaker)

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Molecular Biology II Transcription Regulation in
Bacteria

Stem: 4-10nts, G-C rich

Loop: 3-8nts

I) Conditional Termination – ρ (rho)

dependent
~50% mRNA termination for protein encoding genes

Not definable by a simple consensus sequence, rho recognises sequence features and structural
features instead
Rho mediates transcript release at a DNA sequence where the transcription complex is too stable
for spontaneous release

Rho binds RUT site in RNA, and moves in the 5’  3’ direction

 Known as a RUT (Rho Utilisation) site – 40bp+ ssRNA containing many C residues
The movement of rho is coupled with ATP hydrolysis (only cleaving the γ-phosphate)
Rho moves faster than RNAP

Once rho reaches RNAP, it uses its ATP driven

helicase activity to unwind the DNA/RNA helix
(does not involve weaker A-U binding)

Rho has a hexameric ring structure

It can unfold, allowing RNA to enter the centre
This allows rho to attach midway through an
RNA molecule, rather than ‘threading through’
the entire strand
The RNA first binds to primary binding sites on
the outside of the molecule then, enters.

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Molecular Biology II Transcription Regulation in
Bacteria

Transcription and translation are coupled in prokaryotes

Not the case in eukaryotes: transcription  modification  splicing  export  translation

The trp operon:

On hand drawn sheet

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Molecular Biology II Transcription Regulation in
Bacteria

Low Trp levels

 Slow translation of leader peptide from domain 1

 Hairpin formation between domains 2 & 3
 Transcription is allowed

High Trp levels

 Fast translation of leader peptide from domain 1

 Domain 2 blocked by ribosome
 Hairpin formation between domains 3 & 4
 Formation of terminator structure

Lecture 18 [Page 5]