Lecture 10 Flashcards

(35 cards)

1
Q

Central Dogma:

A

DNA into RNA into Protein into Function

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2
Q

The Problem at Hand

A

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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3
Q

Simple Definitions

A

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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4
Q

Bacterial genes can be clustered in Operons

A

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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5
Q

Regulons

A

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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6
Q

Promoters

A

Region of DNA that control transcription of adjacent genes

RNA Polymerase binds to promoter to start transcription

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7
Q

Features of E.Coli Promoter

A
  • 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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8
Q

Features of E.Coli Promoter (2)

A

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

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9
Q

RecA promoter

A

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

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10
Q

araBAD promoter

A

controllers arabinose utilization operon is weak promoter

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

Weak Promoter

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11
Q

Promoter Combination

A

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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12
Q

Bacterial RNA Polymerase (RNAP)

A

Is a holoenzyme composed of several subunits

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13
Q

RNAP Alpha Subunit

A

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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14
Q

RNAP Beta and Beta’ Subunits RNAP Omega Subunit

A

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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15
Q

RNAP Sigma subunit

A

Binds to promoter

Targets RNAP to correct sequence on chromosome

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16
Q

Core Promoter Elements

A

Are recognized by different RNAP domains

-35 and-10 regions bind to specific domains of sigma

Up element binds to CTD of Alpha

17
Q

Bacterial Transcription - Step 1 Promoter Recognition

A

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

18
Q

Bacterial Transcription - Step 2 Isomerization

A

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

19
Q

Domain 2 of Sigma subunit

A

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

20
Q

Bacterial Transcription - Step 3 Initiation

A

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
21
Q

Bacterial Transcription - Step 4 Promoter escape and Elongation

A

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

22
Q

Bacterial Transcription - Step INFO

A

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
23
Q

Basic Mechanisms of Regulation

A

Alternative sigma factors

Transcription factors

Small Ligands (cAMP, ppGpp)

Local chromosome structure (Super coiling folding)

24
Q

RNAP core

A

can change to different sigma factors to bind to different promoters

25
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
26
Different Sigma Factors regulate Genes Important for Different Functions
27
Different Sigma Factors have different consensus promoter sequences
28
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
29
Simple Activation: Class 1 activated promoters
DNA binding proteins target specific seq upstream of promoter Help recruit RNAP to suboptimal promoter by binding CTD
30
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
31
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
32
Simple Repression: blocking RNAP from binding its promoter
Repressor binds to operator seq within promoter Blocks access to RNAP
33
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
34
Less Simple Repression
Modulating the activity of an activator
35
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