Biochemistry 441 Lecture 6 Ted Young February 16, 2000

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(No Transcript)
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Exam Monday covers lectures 1-10.Remember
Examinations are formidable even to the best
prepared, for the greatest fool may ask more than
the wisest man can answer.

• Charles Caleb Colton-British clergyman and
  writer,
• ?1780-1832

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Biochemistry 441Lecture 11Ted YoungFebruary 1,
2008

• Todays topic Transcription coupling genotype
  to phenotype.

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The Holy Trinity of molecular biology DNA makes
RNA makes Protein
1. Mechanism
2. Control
XXXXX
Reverse transcription (RNA viruses, telomerase)
RNA replication
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Transcription

• What is a gene? How do we identify genes in
  biochemical/molecular terms? Three common ways
  (none foolproof)
• 1. DNA sequence gtopen reading frames (ORFs) (but
  introns genes encoding rRNA, tRNA, siRNA,
  snRNA). Genetic analysis mutations.
• 2. RNA analysis gtexperimentaltranscribed region
  of the gene (hybridization to an oligonucleotide
  array is best).
• 3. Binding analysis gtin vitro and in vivo
  binding assaysgtregulatory regions

Genetically a gene is defined as a region of DNA
that controls a discrete hereditary characteristic
. The complete gene contains protein coding
(exon) and non-coding (intron) DNA that is
transcribed, as well as non-transcribed
regulatory DNA.
top strand
DNA non-template (coding or sense) strand
5 3
3 5
transcribed region
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Mapping the 5 end of RNA and determining which
strand is transcribed
DNA
5-P
RNA
1. S1 nuclease mapping
5-P
Restriction enzyme digestion. Purify fragments
D to denature
Anneal 65oC with RNA
3-end of DNA sequence corresponds to 5 end of
RNA
D to denature
Sequence DNA
5-P
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2. Primer extension
3. DNA microarrays-how could you use DNA
microarrays to map the 5 end of RNA and
determine which strand is transcribed?
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Measuring RNA levels in cellshttp//pathmicro.med
.sc.edu/pcr/realtime-home.htm
Problem there are 30,000 different RNAs, each
present in a different amount in each human cell.
Need a technique that is both very sensitive and
very specific to detect and measure each RNA.
Quantitative reverse transcriptase real-time
PCR is the best method.
Copy with reverse transcriptase (RT) using short
(9 nucleotide), random (NNNNNNNNN) primers ( )
Isolate total RNA from cells
real-time PCR using a pair of primers( )
for the gene being assayed
PCR in the presence of a dye that fluoresces when
it is bound to DNA measures product
accumulation as a function of time. You can see
from the graphs that this method is sensitive
and quantitative over a 10e8-fold range!
Plotting amount of DNA on a linear or a log scale
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RNA synthesis

• 1. Bacterial RNA polymerase structure and
  function.
• 2. Sigma subunit and specific DNA binding at
  promoters
• 3. Initiation, elongation, and termination of RNA
  synthesis.

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Conserved features of RNA polymerizing
enzymes-RNA polymerases

• 1. Couple 5-NTPs to make an RNA chain.
• 2. Copy one strand of template DNA (which one?).
• 3. Initiate an RNA chain de novo (no primer
  required).
• 5NTP

DNA, Mg
RNA PPi
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Structure of RNA polymerase from Thermus aquaticus
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Transcription process
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RNA polymerases subunit composition

• Types of RNA polymerase enzymes Size
• T7 bacterial virussingle polypeptide chain. 99kD
• Bacterial five polypeptide chains. 450kD
• Eukaryotic three nuclear enzymes, each
  containing
• 10-12 polypeptide chains 600kD

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Steps in transcription

• 1. Binding
• Non-specific gt specific
• 2. Initiation
• 3. Elongation
• 4. Termination and release

DNA template

RNAP
Template strand
ATP, GTP
3
5
NTP
5
RNA
5
3
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Binding

• Binding consists of three steps
• 1. Random binding to DNA
• 2. Finding apromoter-sequence in the DNA where
  RNAP binds strongly and initiates transcription.
• 3. Melting the promoter
• (temperature-dependent)

drifting
P
Promoter binding
P
Protected region
10-12 bp melted
bases sensitive to oxidation in melted region
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Determining binding site for proteins on DNA
32P
()

• 1. DNase footprinting

protein

DNase





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DNase footprinting (cont.)

(-)





foot-print

• Remove protein, run sequencing gel

()



Protected from digestion
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Promoters

• Promoters are specific DNA sequences that
  determine where and how often transcription
  initiation occurs

RNA
met
ATG
Bacterial promoter consensus sequence
-35 -10 1
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Role of sigma in promoter recognition
s

• Bacterial RNA Pol subunits composition s, b,b,
  a(2). w
• Core, b b a2, w, binds to DNA in the absence
  of a promoter but only initiates at nicks and
  gaps, and transcribes non-specifically.
• is responsible for specific initiation
• Bacteria contain multiple sigma factors, each
  endowing the holoenzyme with the ability to
  recognize a different promoter sequence.
• There are unique sigma factors for different
  classes of genes those that allow the cell to
  respond to high temperature, to nutrient
  deprivation, etc. Each one allows the RNA
  polymerase to transcribe genes whose protein
  products are important for growth and survival
  under different conditions.

s CYCLE
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Sigma and specific initiation
RNA pol activity on template DNA
single- stranded T4 Crude
extract 100 100 Fraction no. 7 0
0 50 0 0 54 100
0 5054 100 100
(total protein content)
Polymerase activity on calf thymus DNA
No pol activity
s
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Sigma and specific initiation

• 1. RNA pol in E. coli crude extracts will
  transcribe both double-stranded phage DNA and
  single-stranded DNA.
• 2. After passing the crude extract over a strong
  ion exchange column, activity on single-stranded
  DNA is recovered in the fraction containing a2,
  b, and b but no s. These fractions had no
  activity on phage DNA which had no nicks or gaps.
• 3. Add-back experiments showed core polymerase in
  the active fractions and sigma was discovered in
  a fraction which would restore activity on the
  phage DNA.
• 4. Inference sigma was required for
  transcription from genuine promoters core would
  suffice for initiation if the DNA was
  single-stranded or contained nicks or gaps that
  created easily melted regions.

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Elongation RNA is synthesized from 5 to 3,
copying DNA in a 3-to-5 direction
The catalytic mechanism is essentially the same
mechanism used by DNA polymerase, and all other
nucleic acid polymerizing enzymes an attack by
the 3OH at the end of the growing RNA chain on
the a phosphate of the incoming NTP.
The template DNA strand is also called the
antisense strand
5 end
3 end
N1 N2 Nx Nx1
5
3
Nascent RNA strand
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Transcription termination
An RNA hairpin followed by a U-rich sequence
causes termination of transcription and release
of the RNA from the template and release of the
enzyme from DNA.
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Transcription by bacterial RNA polymerase-summary

• One enzyme synthesizes all RNAs in a bacterial
  cell-messenger, ribosomal, and transfer RNA (but
  not in eukaryotes-as we will see).
• Transcription initiates at specific sites called
  promoters and requires sigma, the specificity
  factor.
• Elongation copies one DNA strand(the antisense
  strand), synthesizing RNA 5 to 3 while copying
  DNA 3 to 5.
• Transcription termination occurs at specific
  sites when a specific stem-loop structure in the
  RNA is synthesized.

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Characteristics of quantitative real-time PCR