Lecture #7

UP Element

Upstream of the core promoter, stimulating transcription by a factor of 30

• Part of promoter because it is recognized by RNA polymerase

Enhancers

Additional cis-elements between -60 and -150 which are bound by transcriptional activators

E.coli sigma factor 70

Sigma factor that contains conserved domains

• 4 regions of high sequence similarity

• Specific areas that recognize the core promoter elements are the -10 and -35 box

Sequence recognition specification is achieved through interaction between specific aa of sigma factors and the promoter sequence

• recognition seals DNA in the polymerase

Temporal (time dependent) control of transcription

At different stages of infection, different genes are turned on

ex: SPO1-infected B. subtitles

1. Early genes turned on in first 5 minutes of transcription by the host holoenzyme

   1. Sigma factor Gp28: early gene that replaces host sigma 43, changing the specificity of RNA polymerase to transcribe middle genes

2. Middle genes, gp33 and gp34, encode proteins that bind to each and forma new sigma factor for late genes

Role of alpha subunit

Recognizes the upstream promoter element

• sigma factor recognizes core promoter element

• 

Gel Mobility Shift

Gel electrophoresis can be used to detect DNA and complexes

notice the different bands identify parts of complexes

DNase foot printing

Detects DNA-protein interaction using the fact that a protein bound to DNA will often protect that DNA from enzymatic cleavage

1. Digest DNA-protein complex under mild conditions with DNase 1 (endonuclease)

2. protein binding prevents the digestion from DNase 1

No bands indicate that DNA is bound to protein; called the footprint

• less bands indicate larger protein concentration

Evidence of alpha binding to Up element

No bands=protein-DNA complex

• Alpha subunits show no bands around Up element, giving evidence that it is bound to the UP element

• RNA Polymerase shows binding at UP element and at the core promoter

Stages of transcription initiation

1. Formation of a closed promoter complex

2. Conversion of closed promoter complex to an open promoter complex

3. Polymerizing early nucleotides

4. Promoter clearance: transcript becomes long enough to forma stable hybrid w/ template

Reuse of sigma factor

During initiation, sigma factor can be recycled for additional use with new core polymerase

• córę enzyme can release sigma factor which is then free to associate with another core enzyme

Elongation

After initiation:

• córę enzyme continues to elongate RNA

• Nucleotides are added sequentially

• Phosphodiester bonds formation involves B and B’ subunits

  • Subunits also involved in DNA binding

Rho-independent termination

Depends on:

• Inverted repeats followed by T-rich region in the nontemplate strand of the gene

  • Forms a hairpin structure due to complementary base pairing between inverted repeat sequences

  • Inverted repeats allows a hairpin to form at a transcript end

  • String of T’s in contemplate strand results in week base pairs holding the transcript to the template strand

Model of Intrinsic/rho-independent Termination

1. base pairing of something to the transcript to destabilize RNA/DNA hybrid

   1. Causes hairpin to form

2. Causes transcription to pause

   1. String of Uc incorporated downstream of hairpin to destabilize hybrid and the RNA falls of the DNA template

Rho-dependent termination

Rho binds to the RNA polymerase in an elongation complex, facilitating the dissociating of the RNA/DNA hybrid, terminating transcription

Green line: the presence of rho causes a decrease in total RNA synthesis

• consistent w/ action that rho terminates transcription

Mechanism of Rho

1. Rho follows the polymerase as transcription continues

2. Catches the polymerase as it pauses at the hairpin

3. Releases transcript from the DNA polymerase complex by unwinding th RNA-DNA hybrid