Central Dogma:
DNA into RNA into Protein into Function
The Problem at Hand
Bacterial genome has 500 to 8000 genes
Only subset can be expressed at a time
Inside cell DNA is tightly packed
How does gene regulation work?
Simple Definitions
Gene: Stretch of DNA in genome that encodes for a protein or RNA
Bacteria and archaea genes lack introns
-no true alternative splicing or spliceosomes
Bacterial genes can be clustered in Operons
Operon: - Unit of genetic material functions in a coordinated manner by means of operator, promoter and 1 or more structural genes
Monocistronic: 1 RNA codes for 1 protein
Polycistronic: 1 RNA codes for 3 proteins
Regulons
Operons around the chromosome that share regulation
Any protein that controls gene expression will control several operons at different locations on chromosome
Promoters
Region of DNA that control transcription of adjacent genes
RNA Polymerase binds to promoter to start transcription
Features of E.Coli Promoter
-10 box (Pribnow Sequence) (A short extended -10 sequence)
-35 hexamer 35nt upstream of nucleotides
Up element rich in AT BP found -40 to -60 (varies)
Transcription starts upstream of protein gene
Features of E.Coli Promoter (2)
-35 and -10 boxes and space between them determine strength of promoter
RecA promoter
Strong promoter since its very similar consensus sequence Has 1 different base and smaller spacer
araBAD promoter
controllers arabinose utilization operon is weak promoter
Not very close to -10 or -35 consensus and suboptimal spacing
Weak Promoter
Promoter Combination
Different elements can be altered and combined
Changes strength of core promoter
EX: Poor -10 seq can be made stronger with very good UP seq
Bacterial RNA Polymerase (RNAP)
Is a holoenzyme composed of several subunits
RNAP Alpha Subunit
Identical Alpha subunit per RNAP
2 Domains on Alpha
N-terminal domain (NTD) interacts with RNAP via beta and beta' subunits
C-terminal domain (CTD) interacts with DNA
RNAP Beta and Beta' Subunits RNAP Omega Subunit
2 Distinct subunits and largest
Carry out catalytic reaction reading DNA into an RNA transcript
RNAP Omega Subunit: Plays little role in transcription
Helps beta-subunits assemble properly
RNAP Sigma subunit
Binds to promoter
Targets RNAP to correct sequence on chromosome
Core Promoter Elements
Are recognized by different RNAP domains
-35 and-10 regions bind to specific domains of sigma
Up element binds to CTD of Alpha
Bacterial Transcription - Step 1 Promoter Recognition
RNAP (R) binds to promoter (P) and form closed complex (RPc)
Step driven by RNAP affinity to promoter sequence
DNA still double-stranded, transcription doesn't start
Bacterial Transcription - Step 2 Isomerization
Promoter unwound near -10 Expose ssDNA around -12 to +2
Step Facilitated by sigma factor action
Converting RPc to RPo requires major conformational change in DNA
Domain 2 of Sigma subunit
Recognizes and unwinds DNA at -10 element all at the same time
Domain 2 has two pockets that accommodate conserved A at -11 and T at 7 on non-template strand
Bacterial Transcription - Step 3 Initiation
First few bases are transcribed RNAP is still at the promoter
Abortive cycling of transcription can occur
- Small transcript of <10 BP is made
- RNAP never leaves promoter
Bacterial Transcription - Step 4 Promoter escape and Elongation
Conformation change occurs RNAP escapes promoter and transcribes adjacent gene
Leaves sigma behind
Now in Elongation complex
transcribes whole gene until it receives termination signals
Bacterial Transcription - Step INFO
Transcription initiation steps are reversible
Rate of forward progress depends on:
1. How well RNAP binds to promoter to form RPc
2. How easily RNAP melts DNA to form RPo
3. How easily RNAP escapes promoter and forms elongation complex
Basic Mechanisms of Regulation
Alternative sigma factors
Transcription factors
Small Ligands (cAMP, ppGpp)
Local chromosome structure (Super coiling folding)
RNAP core
can change to different sigma factors to bind to different promoters
Alternative Sigma Factors
E. Coli 7 different sigmas
Each have an optimal target promoter consensus sequence
Diversity in # and types of Sigma between different bacteria species
Some encode 60 sigmas other only have 1 housekeeping sigma
Different Sigma Factors regulate Genes Important for Different Functions
Different Sigma Factors have different consensus promoter sequences
Transcription Factors
Seq-specific DNA binding protein target major groove
Cavity wide enough to accommodate an alpha helix DNA
H-bonds more exposed P backbone and minor groove serve as binding site for protein
Structural differences including width of minor group determine specificity when it's involved
Simple Activation: Class 1 activated promoters
DNA binding proteins target specific seq upstream of promoter
Help recruit RNAP to suboptimal promoter by binding CTD
Simple Activation: Class 2 activated promoters
Protein bind upstream of -35 to contact domain 4 of sigma
Helps RNAP bind to suboptimal core promoter like class 1
Simple Activation: Protein-induced conformational changes in the DNA
Some promoter bind to RNAP poorly
-35 and -10 are not oriented/spaced properly
Some protein improve RNAP binding but bending DNA
Simple Repression: blocking RNAP from binding its promoter
Repressor binds to operator seq within promoter
Blocks access to RNAP
Simple Repression: Generating Looped DNA
Some Repressors trap promoter into a loop
Prevents RNAP binding
Traps RNAP into a complex that cannot escape the promoter
Less Simple Repression
Modulating the activity of an activator
More Complex Arrangements
Combing more than 1 regulator protein at 1 promoter allows better control of transcription in response to environment
Lac Operon is One of them FUCK IT IM TIRED