Clase2 2014 BIOQ400

AMZarraga_BIOQ400_2014 27-04-14
AMZarraga_BIOQ400_2014
27-04-14
Principal steps during DNA transcription
!

RNA POLYMERASE : THE HOLOENZYME
THE TRANSCRIPTION INITIATION CYCLE
2- DOMAIN- CODING STRAND INTERACTION

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I II: required for DNA binding and assembly.
DNA binding and catalysis.
stabilises binding.
forms holoenzyme.

Estab. El RNA
Desestabiliza el hbrido DNA-RNA
DNA se desliza por el lado
NTPs
INTERACTION
RNA POLYMERASE PROMOTER.

Model of the E. coli RNAP (70 2) open complex RP

.
DNA NON CODING STRAND
DNA TEMPLATE
Vista hacia el sitio activo
Non conserved domain
DNA TEMPLATE
RPc
RPo
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! BINDS

!2 BINDS -10

DNA: denatures

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RNA polymerase passes through several
step prior to elongation. Close to open.
Mg++
Sigma Factor
RNA pol Interactions that contributes to promoter recognition
RNA polymerase structure
DNA is forced to make a turn at the active sitedenatures
RNA pol moves remaking bonds..
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non template DNA - " y !70: interactions
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aa DNA contacts

Sunstitution of 70 by an alternative
sigma causes RNA pol to recognize a
different promoter
!
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Figure 7-43 Molecular Biology of the Cell ( Garland Science 2008)
Exchange of during Bacills subtilis infection

RNA pol transcribes
gen 28
Gen28 codes for
Needed to express
34, for the
expression of
middle genes
34 allows the
expression of late
genes

Figure 28.6. Alternative Promoter Sequences.
A comparison of the consensus sequences of standard promoters, heat-shock
promoters, and nitrogen-starvation promoters of E. coli. These promoters are
recognized by s70, s32, and s54, respectively.
!
54
!
70
!
32
TRANSCRIPTION ELONGATION
COMPLEX, TEC
9bp DBS
7-8 bp HBS
Electrostatic and VDWaals
NTPs
Nudler E., Annu. Rev. Biochem. 2009. 78:33561
~9bp
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RNA loop transcription termination
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Termination
Intrinsic terminators are composed of a stable RNA hairpin that can extend to within 8 nucleotides of the 3 end of the RNA
and disrupt the upstream edge of the RNADNA hybrid.
Transcription elongation protein NusA interacts with the nascent RNA near the exit channel and can stimulate termination. Rho
dependent terminators: Rho binds at rut sequence (orange) encoded in the nascent RNA. Rho encircles the RNA and translocates
to the RNA pol. NusG binds Rho and stimulates its activity

Termination and antitermination: RNA polymerase runs a stop sign
Thomas J. Santangelo & Irina Artsimovitch
Nature Reviews Microbiology 9, 319-329 (May 2011)
NusG Elongation Factor (NTD ) interacts withsubunit inhibits backtracking of
RNA pol
assist Rho transcription dependent termination
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I II required for DNA binding and assembly.
needed for DNA binding (contains basic amino acid residues) and catalysis.
stabilises binding.
forms holoenzyme.

Estab. El RNA
Desestabiliza el hbrido DNA-RNA
DNA se desliza por el lado
NTPs
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Bacterial RNA : 5 subunits
T7 Phage DNA: 1 monomer
http://www.wehi.edu.au/education/wehitv/dna_central_dogma_part_1_-
_transcription/

http://www.wehi.edu.au/education/wehitv/
restriction_enzyme_ecor1/
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The promoter: signals RNA pol to bind DNA to
initiate transcription at +1
-10 region
RNAP binds a region of DNA from -40 to +20
The sequence of the non-template strand is shown
TTGACA16-19 bp... TATAAT
-35 spacer -10
Prokaryotic promoter.
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Positive and negative transcription regulation
Molecular signal causes dissociation TF
Molecular signal causes binding
Promoter flanked by regulatory sequences
OPERON
Transcription
! Highly transcribed genes versus poorly transcribed genes.
Focus: Binding of RNA polymerase and isomerization.
! Binding of sigma to RNA pol
! Sigma assures the recognition of PROMOTER, locates
RNA pol at the promoter to initiate transcription in +1
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The function of some mRNAs is regulated by small RNAs in Cis or in Trans:
A separate RNA molecule may bind to the mRNA and affects its activity.
This is in Trans

Alternatively, a portion of the mRNA itself may regulate its own function. This
is in cis. A part of the same molecule regualates the function of the molecule.
Cis-regulator:
a sequence of DNA that control the expression of and adjacent gene.

Trans-regulator:
a protein coded somewhere else that diffuses and binds the regulatory sequence of a
gene, or binds at an RNA pol domain or at a regulatory factor located at the
transcription complex.
The Cis and Trans concepts
Allostery regulation
Allostery a mechanism of gene activation
Annu. Rev. Microbiol. 2012. 66:12552
Activating Transcription in Bacteria

Annu. Rev. Microbiol. 2012. 66:12552
Activating Transcription in Bacteria
TF- DNA combinatorial possibilities
Anti- blocks sigma binding to the RNA pol, another strategy of gene regulation
GENE REGULATION OF OPERONS
NOBEL AWARD: JACOB- MOND - WOFF
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.
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When lactose is added to the culture as a source of glucose, galactosidase is synthesized
Allolactose is the inducer of the operon
upon its binding to the repressor..
!
Bacterial Operons Are Coregulated Gene Clusters
How do bacteria adapt so quickly to their environments?

Part of the answer lies in clusters of coregulated genes called operons.
Allolactose binds
Lac operon contains 3 operators: - 90 bp, down
promoter and 400 bp downstream.
Lac repressor binds as a dimer to each operator
Trp operon regulation: negative regulation
" CTD: C-terminal domain of the " subunit of RNAP
Catabolic regulation: cAMP dependent Lac operon regulation.

CAP binding site is 60 bp upstream of the start site of transcription.
CAP interacts with the RNAP and recruit it to the promoter.
The LAC operon
Activation upon
binding of CAP
The LAC operon
recognition helix fits into the major groove of the
DNA.
CAP and Lac repressor bind DNA using a common structural motif: HTH
motif ( helix-turn-helix )
" CTD
Interaction CAP RNA Polymerase
CAP binds as a
dimer
DNA binding Domain
Activation of Glutamine A gene expression, dependent on Nitrogen deprivation:
through the formation of a gene activator loop

Exposure to low N level:
-E. Coli TF NtrC binds at 150 as a dimer
I -intereact with 54 and activate gene transcription
CAP site
194 bp
AraC and CAP activators activate the araBAD operon expression
Tucker1 and Breaker, Current Opinion in Structural Biology 2005, 15:342348
The 2nd structures of 7 riboswitches
and metabolites that they sense

Clase2 2014 BIOQ400

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