BIO 504 - Prokaryote Genome III

54

sigma ___ stimulates transcription and expression of glutamine synthetase glnA in N regulatory pathway

• Needed for arginine catabolism

• NO ability to melt DNA on its own unlike other sigma factors

NtrB

One of the 2 proteins (antisigma factors) regulating sigma 54

• OFF when glutamine is high aka sensor domain and histidine kinase transmitter domain folded so self-phosphorylation is impossible

• ON when glutamine is low aka sensor domain comes of/unfold and allows self-phosphorylation of histidine kinase transmitter domain

NtrC

one of the 2 proteins (antisigma factors) regulating sigma 54 based on level of glutamine in the cell

• OFF the regulatory domain is bound to DNA, preventing the other domain to bind to it

• ON the DNA-binding domain is bound to DNA, because the phosphate from the histidine kinase transmitter domain is transmitter to the regulatory domain allowing the binding and transcription

silent complex

sigma 54 and RNA poly are bound to DNA, but NOT to NtrC

• CANNOT transcribe

active complex

sigma 54 and RNA poly are bound to DNA AND to NtrC

• initiate transcription

anti-sigma factors

proteins regulating sigma factors by EITHER marking them for degradation OR sequestering them by binding to them so they block their association with RNA poly

• sigma 54 is the ONLY one without any ___ because it doesn’t need it as it cannot melt DNA without NtrC

HseC, Rsd

___ and ___ are the anti-sigma factors of sigma 70

HscC

heat shock chaperone

anti-sigma factor of sigma 70

• sequester it

Rsd

Regulator of sigma D

anti-sigma factor for sigma 70

• sequester it

RseA

Regulator of sigma E

anti-sigma factor of sigma 24 which deals with peroxidative extracellular stress

• sequester it under normal conditions btu release it in response to stress

FlgM

flagella protein M

anti-sigma factor for sigma 28 which deals with flagella genes

• sequester it

DnaK

heat shock chaperone

anti-sigma factor for sigma 32 which deals with heat shock response

• sequester it

Rssb

regulator of RpoS

anti-sigma factor for sigma 38 dealign with stress response and maintaining stationary phase

• marks it for degradation

own expression

sigma 70 regulates its ___ meaning that there is always a small amount of sigma 70 available in the cell despite anti-sigma factor, and also why the regulation mechanism is sequestration and not degradation.

anti-anti-sigma factors

___ are proteins regulating the anti-sigma factors

+1

where is the transcriptional start site of a gene?

5’ race techniques, empirically

How would you identify the transcriptional start site of a gene?

-10, -35, up elements

what are the transcriptional control elements?

look at consensus sequence and see which one matches with promoter of gene of interest

How do you determine which sigma factor needed for transcription?

downstream of transcriptional start site

Where is the translational start site (RBS) relative to promoter?

No, it doesn’t recognize stop codon

Does RNA poly fall off when reaching a stop codon?

expensive

transcription is generally ___, and thus remain the main regulation of gene expression in both prokaryotes and eukaryotes

abortive transcription

After binding to sigma factor, recognizing the promoter, and isomerizing to open complex, RNA poly starts transcribing but stops and falls off after 5-7 nucleotides

• sigma factor holding it back by its link to -10 and -35 elements?

Transcription steps

1. Holoenzyme binds to sigma factor

2. Promoter is recognized and complex binds DNA

3. Close complex formation

4. Isomerization to open complex formation

5. Transcription initiation

6. Promoter escape: sigma factor falls off and replaced by a protein

7. Elongation

8. Termination

transcription bubble

region of duplex DNA in open complex where it is separated

• about 17bp in size

sigma 4

domain of sigma factor binding to -35 element

sigma 3, sigma 4

___ and ___ are domains of sigma factor binding to the -10 element

negative

__ supercoiling are BEHIND the RNApoly enzyme during elongation

• the strain created induces DNA melting and makes melting the start site easier for the next RNA poly

positive

__ supercoiling are AHEAD of the enzyme

RNA-DNA duplex

During elongation, in the transcription bubble, a ____ forms, with the newly made RNA attached to it as a part of it, which anchors the enzyme to the DNA and adds stability