8/12/2019 Molecular Biology Lecture 4
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BIOL321 - Transcription
Cristofre Martin
Department of Biochemistry
St. George’s University
8/12/2019 Molecular Biology Lecture 4
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There are two major steps that occur during protein
synthesis:
a) Transcription - the synthesis of a single-stranded RNA copy
of a segment of DNA.
b) Translation - the conversion of the messenger RNA base
sequence information into the amino acid sequence of a protein polypeptide.
The “Central Dogma” of Molecular Biology
DNA RNA ProteinTranscription Translation
Reverse transcription
Replication
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Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
The Basic Transcription Process
1. The DNA unwinds in a short region next to a gene.
2. RNA polymerase catalyzes the synthesis of an RNA
molecule in the 5’ to 3’ direction along the 3’ to 5’ template
strand of the DNA.
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Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
RNA polymerase
catalyzes thesynthesis of an
RNA molecule in
the 5’-to-3’
direction along the3’-to-5’ template
strand of the DNA.
Ribonucleoside
triphosphates are
the RNA precursor
molecules used for
RNA synthesis.
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01_12.jpg
During transcription only one strand of DNA is required as a template.
There is evidence that genes can be transcribed from any strand.
The strand serving as the template for transcription is called the
antisense strand and the other strand is called the sense strand (same
sequences as the RNA)
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Comparison of DNA synthesis and RNA synthesis:
RNA DNA
RNA polymerase DNA polymerase
NTPs precursor dNTP precursor
no primer needed primer required for initiation
uracil pairs with adenine thymine pairs with adenine
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There are four different types of RNA molecules:
1) mRNA (messenger RNA) encodes the amino acidsequence of a polypeptide. mRNAs are the
transcripts of protein coding genes.
2) tRNA (transfer RNA) brings amino acids to the
ribosome during the translation process.3) rRNA (ribosomal RNA) combines with ribosomal
proteins to form the ribosome. mRNA is translated
into protein at the ribosome.
4) snRNA (small nuclear RNA) combines withcertain proteins and is involved in RNA processing
(example mRNA splicing) in eukaryotes.
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In bacteria, there is only a single RNA polymerase:
-composed of the core polymerase plus sigma factor
There are three different RNA polymerases in eukaryotes:
1) RNA polymerase I: 5.8S, 18S, and 28S rRNA genes
2) RNA polymerase II: all protein-coding genes (mRNA) and
some snRNAs
3) RNA polymerase III: tRNA genes and some snRNAs
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Regions of a prokaryotic gene:
1) Promoter: located upstream of the RNA coding sequence, and
ensures the proper location of transcription initiation.2) RNA coding sequence: the DNA sequence that is transcribed
into RNA.
3) terminator: a sequence downstream of the RNA coding
sequence and specifies where transcription will stop.
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The prokaryote promoter sequences are generally found at -35
and -10 bp from the transcription start site.
-35 consensus sequence: 5’-TTGACA-3’ -10 consensus sequence: 5’-TATAAT-3’ (also called the Pribnow
box)
***Variation within these sequences results in variation in the binding ability of RNA polymerase and can affect the rate of
transcription.
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Initiation and elongation of the RNA transcript in E. coli.
a) RNA polymerase
(holoenzyme - core
RNA polymerase
plus sigma factor)
binds to -35 and -10
regions.
b) DNA molecule is
unwound by
approximately 17
bp in the -10 region.
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c) After 8 or 9
ribonucleotides
have been
polymerized thesigma factor
disassociates with
the core enzyme.
d) Polymerization proceeds as RNA
polymerase
unwinds DNA
molecule.
e) Transcription is
terminated once a
termination
sequence is reached.
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Eukaryotic promoters consist of a collection of conserved short
sequence elements located near the transcription start site:
Cis element DNA sequence Approx. Position
GC box GGGCGG -70 to -200
TATA box TATAAA -20 to -35
CAAT box CCAAT -80
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Transcription Initiation in Eukaryotes Requires Many Proteins:
1) TATA box binding protein
-a subunit of TFIID-binds to TATA box of gene promoter
2) TFIID - causes a distortion in the DNA helix allowing the
recruitment of other transcription factors.
3) TFIIB - involved in RNA polymerase interactions
-start site recognition
4) TFIIH
- contains a DNA helicase to unwind DNA
-activates RNA polymerase by phosphorylation
5) TFIIE - involved in positioning RNA polymerase
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Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Transcription initiation in eukaryotes:
1) The most common
eukaryote promoter
sequence is located at position -25 and
contains the
consensus sequence:
5’-TATAAAA-3’
(TATA BOX).
2) TBP/TFIID bind to
the promoter at the
TATA box (-25).
3) TFIIB binds to
promoter.
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4) RNA polymerase, TFIIE, and
TFIIH are recruited to the
promoter and transcriptioninitiation site.
5) TFIIH unwinds the DNA.
6) TFIIH phosphorylates RNA
polymerase activating it for
transcription.
7) Most transcription factors arethen released from the basal
transcriptional machinery
prior to transcription
initiation.
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The RNA pol II and the transcription
factors TFIID (TAFs and TBP), and
other transcription factors form the
basal transcriptional machinery.
The basal transcriptional machinery
is sufficient for only a low level oftranscription.
For increase levels of transcription or
tissue specific transcription, the binding of an activator and adaptor
molecule is required.
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Transcription of DNA results in positive (+ve) supercoils in front
of the RNA polymerase and negative (-ve) supercoils behind the
RNA polymerase.
Transcribing DNA Overwound
(+ve supercoils)Underwound
(-ve supercoils)
Gyrase
Introduces -ve
supercoils
Topoisomerase
Relaxes -ve
supercoils
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Termination of transcription can involve intrinsic termination
sequences found in the RNA transcript.
1) Palindromic regions that form hairpins varying in length from 7
to 20 bps.
2) The stem loop structure includes a G-C rich region and is
followed by a run of U residues.
G C
C
A
AA
G
C
C
C
U
A
A
G
U
UU
C
G
G
G
A
U
U
U U U U
A G
U
G
G
CC
G
U
A
G C
A
G
U
AG
G-C rich region in stem
Single-stranded U run
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Termination of transcription can involve extrinsic factors such as
the rho protein.
1) Rho protein attaches to
recognition site on the RNA.
2) Rho moves along RNA following
RNA polymerase.
3) RNA polymerase pauses at
terminator and rho catches up.
4) Rho unwinds DNA-RNA hybrid
and pull RNA from the RNA polymerase.
5) Termination: RNA polymerase,
rho, and RNA are released.
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Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
General structure of mRNA found in bothprokaryotic and eukaryotic cells
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Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
In eukaryotes, the
initial produce of
transcription must
be processed and
modified to form
the mature mRNA.
1) Pre-mRNA is
spliced to remove
introns (non protein
coding regions).
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Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Cap structure at the 5 end of a eukaryotic mRNA
2) The addition of a 7-methylguanosine to the 5’ end of the
mRNA.
This process is called 5’ cappingand is necessary for the
binding of the mRNA to the
ribosome which is essential for
the translation process.
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3) The addition of
a poly-A tail tothe 3’ end of
the mRNA.
The poly-A tail is
necessary for
mRNA
stability.
Polyadenylation of mRNA
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Fidelity of RNA polymerase
• RNA polymerase lacks the proof reading activity that is found
in DNA polymerase.
• the accuracy of transcription is much less than that of
replication.
• error rate is about 1/10,000 base pairs
• this level of error is tolerated in the cell because there many
transcripts from each gene and most will be error free
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