Regulation of Gene Transcription ( I,

Prokaryotes)

•Regulation of gene expression lead to differential gene expression in different cell types or developmental stages or in response to external conditions.

•RNA polymerase initiate transcription, for synthesizing mRNA.

•In prokaryotes, the newly formed RNA transcripts is functional mRNA without further modification .

Regulation of gene expression

DNA

RNA

Protein

Transcription

Processing &

splicing

Translation

An Overview of Transcription

Elongation

Termination•RNA polymerase recognizes terminator seq. or require another ρ(rho) factor

Transcription unit :contain the DNA between promoter, where transcription initiates, and a terminator, where it ends.

E coli RNA Polymerase RNA polymerases :are large proteins which contain

core polymerases: α2ββ 'ω (composed of β, β‘, two α and one ω subunits).

holoenzyme: a core polymerase plus σfectors

α subunits: promoter binding .

β subunit: involved in both transcription and elongation.

β’ subunit: involved in template DNA binding

ω subunit: stabilized core polymerases structure

Sigma(σ) factor: required at the initiation stage for recognizing

the promoter.

Transcription - initiation

•Promoter binding

•DNA unwinding

•RNA chain initiation

•RNA chain elongation

Transcription termination I•Intrinsic terminators contain a GC-rich hairpin followed by a run of U residues •They are recognized in vitro by core enzyme alone.

Transcription termination II ---ρ(Rho) dependent terminators •ρ factor is a hexameric protein hydrolyzed ATP in the presence of single-strand

RNA•recognized a RNA stretch of 50-90 nucleotides preceding the site of termination that

is rich in C and poor in G residues. •ρ factor is an essential protein that acts as an ancillary termination factor, which

recognizes RNA and acts at sites where RNA polymerase has paused.

Regulation of transcription in prokaryotes

• Regulatory proteins contain structure motifs that can read DNA sequence.

• Footprinting and gel-shift assays identify protein-DNA interaction

• The helix-turn-helix motifs one of the simplest and most common DNA-binding motifs

• Positive control • Negative control

λ-phage gene expression studies

Phage sequence-specific RNA-binding proteins can regulate termination by E. coli RNA polymerase

Antitermination can be used to control transcription

λ phage lytic cycle development pathway

Immediate early gene product: N protein induces antitermination

N-protein induces antitermination

Lambda immediate early and delayed early genes are needed for both lysogeny and the lytic

cycle

• Lambda has two immediate early genes, N and Cro, which are transcribed by host RNA polymerase.

• N is required to express the delayed early genes.• • Three of the delayed early genes are regulators.

• Lysogeny requires the delayed early genes cII-cIII.

• The lytic cycle requires the immediate early gene cro and the delayed early gene Q.

The phage λmap shows clustering of related function.

The genome is 48,514 bp

Antitermination is used by phage to regulate progression life stage in bacteria

• pN (immediate early) is an antitermination factor that allows RNA polymerase to continue transcription past the ends of the two immediate early genes.

• pQ is the product of a delayed early gene and is an antiterminator that allows RNA polymerase to transcribe the late genes.

• Because lambda DNA circularizes after infection, the late genes form a single transcription unit.

Lysogeny requires several events

cII/cIII cause repressor synthesis to be established and also trigger inhibition of late gene transcription.

Establishment of repressor turns

off immediate and delayed early gene expression.

Repressor turns on the maintenance circuit for its own synthesis.

Lambda DNA is integrated into the bacterial genome at the final stage in establishing lysogeny.

The Cro repressor is needed for lytic infection

Cro binds to the same operators as repressor but with different affinities.

When Cro binds to OR3, it prevents RNA polymerase from binding to PRM, and blocks maintenance of repressor.

When Cro binds to other operators at OR or OL, it prevents RNA polymerase from expressing immediate early genes, which (indirectly) blocks repressor establishment.

What determines the balance between lysogeny and the lytic cycle

The delayed early stage when both Cro and repressor are being expressed is common to lysogeny and the lytic cycle.

The critical event is whether cII causes sufficient synthesis of repressor to overcome the action of Cro.