Prokaryotic Transcription Flashcards
Describe biochemistry of prokaryotic transcription
Incoming phosphates are named alpha, beta, and gamma (beta and gamma leave after attachment
RNA builds 5’-3’, DNA template is 3’-5’
You can replace gamma or alpha phosphate with a labeled phosphate
Promoter Consensus Sequence
-35 and -10. they are named in reference to their location in relation to the start site. -35 is higher in C-G, whereas -10 is high in A-T which makes it easy for RNA polymerase to separate DNA strand
Footprinting
Identifies DNA-binding sites for protein by their protection against nicking
RNA Polymerase composition
Holoenzyme: 2-alpha, 2-beta, 1-sigma
Core: 2-alpha, 2-beta
Early transcription events
- form the closed promoter complex, core plus sigma makes holoenzyme
- form the open promoter complex
- incorporate 1st few nucleotides
- promoter clearance, sigma leaves
- elongation
Properties of Sigma
- Confers promoter specificity
- Stimulates tight DNA binding
- Stimulates initiation
- Can be reused
sigma-factor tread-milling
Once the 1st 10 bases are transcribed, sigma releases and attaches to new core to create new holoenzyme. Leads to multiple RNA polymerases transcribing at once. Produces christmas tree effect
Torsional stress
Gyrase un-winds DNA strand at leading edge before going into polymerase.
Topoisomerase re-winds DNA strand following transcription.
Transcription termination
Rho independent
Rho dependent
Rho independent transcription termination
A sequence of inverted repeats (C-G rich) leading up to a multi-T sequence causes formation of a hairpin which leads to de-stabilization of the enzyme interaction
Rho dependent transcription termination
- Decreases net rate of RNA synthesis
- Reduces size of RNA products
- Releases RNA products from DNA
Rho binds to transcript at rho loading site, pursues polymerase, hairpin forms, polymerase pauses, rho catches up, rho releases transcripts and causes termination
Common gene regulation themes
- Selective usage of promoters is mediated by the binding of protein to specific DNA sequences( - proteins turn genes off, + proteins turn genes on)
- Promoter binding influences promoter utilization by:
a. Blocking the binding or movement of other proteins
b. Promoting the binding of other proteins
c. Interacting with basal transcription apparatus - Interaction of regulatory proteins with DNA must be reversible and accessible to regulation
- Coordinated regulation of gene expression can be accomplished by propagation of common binding sites for regulatory proteins
General features of regulated gene expression unique to prokaryotes
- Transcription and translation are coupled
- Genes can be arranged in operons
- The half life of mRNA is normally on the order of minutes
General features of regulated gene expression unique to eukaryotes
- Gene accessibility - chromatin
- Transcript Transport/Localization
- Multicellularity
Lactose utilization by E. coli
Lactose is brought in through a permease, beta-galactosidase converts to galactose and glucose (fuel for cell)
Lac operon organization
6000bp total
- Promoter
- lacI - Repressor, 1040bp
- Operator, 82bp
- lacZ - beta-galactosidase, 3510bp
- lacY - Permease, 780bp
- lacA - Transacetylace, 825bp
Lac operon description
- lacI makes mRNA that makes repressor protein
- Repressor binds to operator and prevents transcription of z, y, and a
- When inducer (lactose) is added it binds to repressor protein, preventing it from binding to operator
- Absence of active repressor allows RNA polymerase to access promoter and transcribe mRNA for z, y, and a proteins
PaJaMo experiment
Addition of lacI and lacZ wild type genes to lacI and lacZ deficient mutants stimulates synthesis of beta-galactosidase for a short time, but lacI eventually represses synthesis. When inducer is added to same model, beta-galactosidase continues to increase.
Problems with Lac Operon Regulation
- Formation of allolactose (inducer) requires β-galactosidase activity
- Entry of lactose into the cell requires the lac permase
- The binding of regulatory proteins to DNA is not infinitely strong
- Coordinate regulation with glucose availability
- Transcription of the Lac operon is inhibited when glucose is present
- Catabolite Activator Protein is a positive acting regulatory protein
CAP
Catabolite Activator Protein is a positive acting regulatory protein
Describe Lac Operon Regulation
- Glucose present (cAMP low); no lactose
a. lacI produces repressor which binds to operator
b. CAP does not bind
c. Gene off, no mRNA - Glucose present (cAMP low); lactose present
a. lacI produces repressor, lactose (inducer) binds to repressor, repressor does not bind to operator
b. CAP does not bind
c. Minimal mRNA - No glucose present (cAMP high); lactose present
a. lacI produces repressor, lactose (inducer) binds to repressor, repressor does not bind to operator
b. CAP binds to promoter, recruits RNA polymerase
c. Abundant mRNA
