Lecture 10 Flashcards Preview

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Flashcards in Lecture 10 Deck (35):
1

Central Dogma:

DNA into RNA into Protein into Function

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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?

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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

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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

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Regulons

Operons around the chromosome that share regulation

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

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Promoters

Region of DNA that control transcription of adjacent genes

RNA Polymerase binds to promoter to start transcription

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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

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Features of E.Coli Promoter (2)

-35 and -10 boxes and space between them determine strength of promoter

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RecA promoter

Strong promoter since its very similar consensus sequence Has 1 different base and smaller spacer

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araBAD promoter

controllers arabinose utilization operon is weak promoter

Not very close to -10 or -35 consensus and suboptimal spacing

Weak Promoter

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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

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Bacterial RNA Polymerase (RNAP)

Is a holoenzyme composed of several subunits

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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

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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

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RNAP Sigma subunit

Binds to promoter

Targets RNAP to correct sequence on chromosome

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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

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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

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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

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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

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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

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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

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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

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Basic Mechanisms of Regulation

Alternative sigma factors

Transcription factors

Small Ligands (cAMP, ppGpp)

Local chromosome structure (Super coiling folding)

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RNAP core

can change to different sigma factors to bind to different promoters

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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

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Different Sigma Factors regulate Genes Important for Different Functions

A image thumb
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Different Sigma Factors have different consensus promoter sequences

A image thumb
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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

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Simple Activation: Class 1 activated promoters

DNA binding proteins target specific seq upstream of promoter

Help recruit RNAP to suboptimal promoter by binding CTD

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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

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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

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Simple Repression: blocking RNAP from binding its promoter

Repressor binds to operator seq within promoter

Blocks access to RNAP

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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

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Less Simple Repression

Modulating the activity of an activator

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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