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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



Operons around the chromosome that share regulation

Any protein that controls gene expression will control several operons at different locations on chromosome



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

A image thumb

Different Sigma Factors have different consensus promoter sequences

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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