Lecture 10

Question 1

Central Dogma:

Answer 1

DNA into RNA into Protein into Function

Question 2

The Problem at Hand

Answer 2

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?

Question 3

Simple Definitions

Answer 3

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

Question 4

Bacterial genes can be clustered in Operons

Answer 4

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

Question 5

Regulons

Answer 5

Operons around the chromosome that share regulation

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

Question 6

Promoters

Answer 6

Region of DNA that control transcription of adjacent genes

RNA Polymerase binds to promoter to start transcription

Question 7

Features of E.Coli Promoter

Answer 7

• 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

Question 8

Features of E.Coli Promoter (2)

Answer 8

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

Question 9

RecA promoter

Answer 9

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

Question 10

araBAD promoter

Answer 10

controllers arabinose utilization operon is weak promoter

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

Weak Promoter

Question 11

Promoter Combination

Answer 11

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

Question 12

Bacterial RNA Polymerase (RNAP)

Answer 12

Is a holoenzyme composed of several subunits

Question 13

RNAP Alpha Subunit

Answer 13

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

Question 14

RNAP Beta and Beta’ Subunits RNAP Omega Subunit

Answer 14

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

Question 15

RNAP Sigma subunit

Answer 15

Binds to promoter

Targets RNAP to correct sequence on chromosome

Question 16

Core Promoter Elements

Answer 16

Are recognized by different RNAP domains

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

Up element binds to CTD of Alpha

Question 17

Bacterial Transcription - Step 1 Promoter Recognition

Answer 17

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

Question 18

Bacterial Transcription - Step 2 Isomerization

Answer 18

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

Question 19

Domain 2 of Sigma subunit

Answer 19

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

Question 20

Bacterial Transcription - Step 3 Initiation

Answer 20

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

Question 21

Bacterial Transcription - Step 4 Promoter escape and Elongation

Answer 21

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

Question 22

Bacterial Transcription - Step INFO

Answer 22

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

Question 23

Basic Mechanisms of Regulation

Answer 23

Alternative sigma factors

Transcription factors

Small Ligands (cAMP, ppGpp)

Local chromosome structure (Super coiling folding)

Question 24

RNAP core

Answer 24

can change to different sigma factors to bind to different promoters

Question 25

Alternative Sigma Factors

Answer 25

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

Question 26

Different Sigma Factors regulate Genes Important for Different Functions

Answer 26

Question 27

Different Sigma Factors have different consensus promoter sequences

Answer 27

Question 28

Transcription Factors

Answer 28

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

Question 29

Simple Activation: Class 1 activated promoters

Answer 29

DNA binding proteins target specific seq upstream of promoter Help recruit RNAP to suboptimal promoter by binding CTD

Question 30

Simple Activation: Class 2 activated promoters

Answer 30

Protein bind upstream of -35 to contact domain 4 of sigma Helps RNAP bind to suboptimal core promoter like class 1

Question 31

Simple Activation: Protein-induced conformational changes in the DNA

Answer 31

Some promoter bind to RNAP poorly -35 and -10 are not oriented/spaced properly Some protein improve RNAP binding but bending DNA

Question 32

Simple Repression: blocking RNAP from binding its promoter

Answer 32

Repressor binds to operator seq within promoter Blocks access to RNAP

Question 33

Simple Repression: Generating Looped DNA

Answer 33

Some Repressors trap promoter into a loop Prevents RNAP binding Traps RNAP into a complex that cannot escape the promoter

Question 34

Less Simple Repression

Answer 34

Modulating the activity of an activator

Question 35

More Complex Arrangements

Answer 35

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