Regulation of Gene Expression in Bacteriophage (lambda)

 

Bacteriophage

>Virus infecting E.coli

>Temperate phage

>50kb ds linear genome in phage

>circular in host

>cos (cohesive ends) site overhangs of 12 bases at 5' ends

>Ligated using DNA ligase and gyrase

>Upon infection, phage can undergo lysis or lysogeny

>Health of host cell determines fate of lambda

Lambda Life Cycle

Lysis

>If cell is healthy, sufficient energy is available for production of progeny phage and lambda phage will go lytic

>3 phases - immediate early, delayed early and late lysis

>production of new phage particles

>replication of lambda phage genome

>production of viral coat proteins

>assembly of progeny phage

>lysis of host cell

Lysogeny

>If cell is unhealthy, lambda will go lysogenic

>3 phases - immediate early, delayed early and late lysogeny

>viral genome integrates into host chromosome passively

>excision of genome from host chromosome by induction when under stress

>phage DNA - prophage

>cell harboring - lysogen

>no progeny produced

>not disadvantageous for host

>Immunity to superinfection (from same phage)

Map of lambda phage

>50 genes coat proteins

>proteins in DNA replication, recombination and lysis

Regulatory Proteins

>Genes clustered into operons

>3 promoters (PR, PL & PRM)

-PR, PL (rightward & leftward)

Strong, constitutive promoters

-Do not need activator proteins

-PRM (Promoter for repressor maintenance)

Weak – directs transcription when activator binds upstream

 2 genes (cI and cro)

>cI - lambda repressor

repress transcription – binds sites that overlap promoter & excludes RNAP

Activate transcription – similar to CAP in activating lac operon; N terminal domain

>Cro (control for repressor and other things)

activate transcription

>Cro and pcI can bind operator sites OR and OL

>operator sites composed of 3 specific operator regions: OR1,, OR2 (overlaps PR, PL), OR3 (overlaps PRM)

Cooperative Binding of cI

pcI 

>2 subunits forming “dumbbell" with DNA binding end and repressor binding end

>forms a dimer to bind DNA

>C terminal provides dimerization contacts and mediates interactions between dimers (tetramerization) – cooperative binding of repressor

>binding affinity: OR1 >>> OR2 = OR3

-binding to OR1 and OR2 is cooperative

-Without cooperativity, 10-fold higher conc of cI needed to bind OR2

Cro

Binds operator in opposite affinity but not cooperatively

cI and cro bind in different patterns

Lysogeny

>low [pcI], binds to both OR1 /OL1 (represses PR /PL) and OR2 /OL2 (activates PRM lambda cI expression)

positive autoregulator

>high [pcI], pcI also binds to OR3 /OL3 - represses transcription of PRM

negative autoregulator

>PR and PL OFF, PRM ON

Lysis

>Cro binds OR3 overlaps PRM represses cI transcription

>RNAP binds PR – transcription of lytic genes

>PR and PL ON, PRM OFF

Prokaryotic vs eukaryotic gene organization

1. Prokaryotic transcriptional regulatory regions (promoters and operators) lie close to the transcription start site
2. Functionally related genes are frequently located near each other in prokayotes
3. These “operons” are transcribed into a single mRNA with internal translation initiation sites

Promoter controls transcription

-Efficiency of recruitment of polymerase determines
   frequency of initiation
-Expression further modulated by regulatory proteins

  

Eukaryotic gene expression regulation by genetic switches is more complex

-Eukaryotic gene transcription is regulated in three ways that are different from those in prokaryotes

    

     "Gene regulatory proteins can control transcription from a distance

     Gene activator proteins promote the assembly of transcriptional complexes

     Packaging of eukaryotic DNA in chromatin"

Eukaryotic pol II promoter

Assembly of initiation complex

-Many steps
-Promoters have different efficiencies


How do promoters differ?
- Promoter strength (recruitment of transcription complex)
- Upstream sequences that bind regulatory proteins

Eukaryotic positive regulation

-Activator works through “mediator” complex
-Activator also modifies chromatin

Inductive Gene Expression

Repressor binds operator site (overlaps with region bound by RNAP)
-no transcription
Absence of repressor, RNA polymerase binds weakly
-basal level expression
Inducer
-Cause production of enzymes to metabolize them
-Removal halts enzyme synthesis (transcription)

Principles of Gene Expression & Regulation

Background
•Cells respond to different factors (environment, heat, food supply, and various external messages)same copy of genome in every cell in an organism, but genes are expressed at different levels in different cells
•Cells differentiate during development
•Gene expression determines what proteins are required at a particular point in development or location
•Gene regulation responsible for dynamic cells occur in different times and places (opening of chromatin, transcription, translation, protein stability, and protein modifications).strongest regulation occurs during transcription

Two types of prokaryotic regulation of transcription

•Negative control: active DNA binding protein represses transcription of the gene(s) downstream
Example: the Trp operon
•When Trp is present in the diet:
–Trp binds to the repressor protein
–This enables it to bind to the promoter
–This binding switches off the genes coding for the enzymes that make Trp
•This makes sense biochemically: it is energetically wasteful to synthesize an AA that is unneeded for bacterial growth
DNA binding proteins can also activate gene transcription

•Positive control: the active form of the DNA binding protein activates transcription downstream
–It is a transcriptional activator
•These proteins help to initiate transcription
–Help RNA pol to bind to the promoter
–Help to initiate transcription of bound, “stalled” RNA pol
Example: the lac operon
•Under both positive and negative control
•Its transcription only occurs if two conditions are met:
–Lactose present
–Glucose absent
•Note that these methods of transcriptional control involve DNA binding protein

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