RNA Polymerase - California Lutheran University

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Transcript RNA Polymerase - California Lutheran University

The Molecular Biology of Genes
and Gene Expression
Central Dogma
• First described by Francis Crick
• Information only flows from
DNA → RNA → protein
• Transcription = DNA → RNA
• Translation = RNA → protein
• Retroviruses violate this order using reverse
transcriptase to convert their RNA genome into
DNA
2
How Genes Work: A Primer
replication (mutation!)
“software” ~ DNA, RNA
genes
DNA
(nucleotides: A,T,G,C)
transcription
RNA
(nucleotides: A,U,G,C)
translation
Protein
(amino acids)
“hardware” ~ proteins
The Nature of Genes
• Early ideas to explain how genes work came
from studying human diseases
• Archibald Garrod – 1902
– Recognized that alkaptonuria is inherited via a
recessive allele
– Proposed that patients with the disease lacked a
particular enzyme
• These ideas connected genes to enzymes
4
Beadle and Tatum – 1941
• Deliberately set out to create mutations in
chromosomes and verify that they
behaved in a Mendelian fashion in crosses
• Studied Neurospora crassa
– Used X-rays to damage DNA
– Looked for nutritional mutations
• Had to have minimal media supplemented to grow
5
• Beadle and Tatum looked for fungal cells lacking
specific enzymes
– The enzymes were required for the biochemical
pathway producing the amino acid arginine
– They identified mutants deficient in each enzyme of
the pathway
• One-gene/one-enzyme hypothesis has been
modified to one-gene/one-polypeptide
hypothesis
6
7
8
• Transcription
–
–
–
–
DNA-directed synthesis of RNA
Only template strand of DNA used
U (uracil) in DNA replaced by T (thymine) in RNA
mRNA used to direct synthesis of polypeptides
• Translation
– Synthesis of polypeptides
– Takes place at ribosome
– Requires several kinds of RNA
9
RNA
• All synthesized from DNA template by
transcription
• Messenger RNA (mRNA)
• Ribosomal RNA (rRNA)
• Transfer RNA (tRNA)
• Small nuclear RNA (snRNA)
• Signal recognition particle RNA
• Micro-RNA (miRNA)
10
Genetic Code
• Francis Crick and Sydney Brenner
determined how the order of nucleotides in
DNA encoded amino acid order
• Codon – block of 3 DNA nucleotides
corresponding to an amino acid
• Introduced single nulcleotide insertions or
deletions and looked for mutations
– Frameshift mutations
• Indicates importance of reading frame
11
12
• Marshall Nirenberg identified the codons that
specify each amino acid
• Stop codons
– 3 codons (UUA, UGA, UAG) used to terminate
translation
• Start codon
– Codon (AUG) used to signify the start of translation
• Code is degenerate, meaning that some amino
acids are specified by more than one codon
13
14
Code practically universal
• Strongest evidence
that all living things
share common
ancestry
• Advances in genetic
engineering
• Mitochondria and
chloroplasts have
some differences in
“stop” signals
15
Prokaryotic transcription
• Single RNA polymerase
• Initiation of mRNA synthesis does not
require a primer
• Requires
– Promoter
– Start site
– Termination site
Transcription unit
16
• Promoter
– Forms a recognition and binding site for the
RNA polymerase
– Found upstream of the start site
– Not transcribed
– Asymmetrical – indicate site of initiation and
direction of transcription
17
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
 Core
 enzyme
9

TATAAT– Promoter (–10 sequence)
Holoenzyme
5‫׳‬
3‫׳‬
Downstream
Start site (+1)
TTGACA–Promoter
(–35 sequence)
Template
strand
Coding
strand
Prokaryotic RNA polymerase
Upstream
5‫ ׳‬3‫׳‬
b.
a.
 binds to DNA
RNA polymerase bound
to unwound DNA
Transcription
bubble
5‫׳‬
3‫׳‬
 dissociates
ATP
Helix opens at
–10 sequence
Start site RNA
synthesis begins
5‫ ׳‬3‫׳‬
18
• Elongation
– Grows in the 5′-to-3′ direction as
ribonucleotides are added
– Transcription bubble – contains RNA
polymerase, DNA template, and growing RNA
transcript
– After the transcription bubble passes, the
now-transcribed DNA is rewound as it leaves
the bubble
19
20
• Termination
– Marked by sequence that signals “stop” to
polymerase
• Causes the formation of phosphodiester bonds to
cease
• RNA–DNA hybrid within the transcription bubble
dissociates
• RNA polymerase releases the DNA
• DNA rewinds
– Hairpin
21
22
• Prokaryotic transcription is coupled to
translation
– mRNA begins to be translated before
transcription is finished
– Operon
• Grouping of functionally related genes
• Multiple enzymes for a pathway
• Can be regulated together
23
24
Eukaryotic Transcription
• 3 different RNA polymerases
– RNA polymerase I transcribes rRNA
– RNA polymerase II transcribes mRNA and
some snRNA
– RNA polymerase III transcribes tRNA and
some other small RNAs
• Each RNA polymerase recognizes its own
promoter
25
• Initiation of transcription
– Requires a series of transcription factors
• Necessary to get the RNA polymerase II enzyme
to a promoter and to initiate gene expression
• Interact with RNA polymerase to form initiation
complex at promoter
• Termination
– Termination sites not as well defined
26
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Other transcription factors
RNA polymerase II
Eukaryotic
DNA
Transcription
factor
TATA box
Initiation
complex
27
Enhancers determine the temporal and
spatial transcription patterns of genes
(drawing modified from Tijan, R., Molecular Machines That
Control Genes, Scientific American 272, Feb, 1995)
Enhancers
Activators = + Auxiliary Transcription Factors
-
Repressor =
auxiliary transcription
factor
Silencer
REPRESSOR
Basal Transcription
Factors
H
E
F
B
A
Co-activators
TATA BOX
RNA Polymerase
CORE PROMOTER
transcription
mRNA modifications
• In eukaryotes, the primary transcript must
be modified to become mature mRNA
– Addition of a 5′ cap
• Protects from degradation; involved in translation
initiation
– Addition of a 3′ poly-A tail
• Created by poly-A polymerase; protection from
degradation
– Removal of non-coding sequences (introns)
• Pre-mRNA splicing done by spliceosome
29
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5´ cap
HO
OH
P
P
CH2
P
+
3´
N+
CH3
Methyl group
P
5´
P
P
mRNA
CH3
30
Eukaryotic pre-mRNA splicing
• Introns – non-coding sequences
• Exons – sequences that will be translated
• Small ribonucleoprotein particles
(snRNPs) recognize the intron–exon
boundaries
• snRNPs cluster with other proteins to form
spliceosome
– Responsible for removing introns
31
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E1
I1
E2
I2
E3
I3
DNA template
Transcription
E4
I4
Exons
Introns
5‫ ׳‬cap
33‫ ׳‬poly-A tail
Primary RNA transcript
Introns are removed
5‫ ׳‬cap
a.
3‫ ׳‬poly-A tail
Mature mRNA
32
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E1
I1
E2
I2
E3
I3
DNA template
E4
I4
Exons
Introns
Transcription
5‫ ׳‬cap
3‫ ׳‬poly-A tail
Primary RNA transcript
Introns are removed
3‫ ׳‬poly-A tail
5‫ ׳‬cap
a.
Mature mRNA
Intron
1
mRNA
3
2
4
DNA
7
5
6
Exon
b.
c.
b: Courtesy of Dr. Bert O’Malley, Baylor College of Medicine
33
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snRNA
Exon 1
snRNPs
Intron
Exon 2
A
5´
3´
Branch point A
1. snRNA forms base-pairs with 5´ end of intron, and at branch site.
34
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snRNA
Exon 1
snRNPs
Exon 2
Intron
A
3´
5´
Branch point A
1. snRNA forms base-pairs with 5´ end of intron, and at branch site.
Spliceosome
A
5´
3´
2. snRNPs associate with other factors to form spliceosome.
35
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snRNA
Exon 1
snRNPs
Exon 2
Intron
A
3´
5´
Branch point A
1. snRNA forms base-pairs with 5´ end of intron, and at branch site.
Spliceosome
A
5´
3´
2. snRNPs associate with other factors to form spliceosome.
Lariat
A
5´
3´
3. 5´ end of intron is removed and forms bond at branch site,
forming a lariat. The 3´ end of the intron is then cut.
36
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snRNA
Exon 1
snRNPs
Intron
Exon 2
A
5´
3´
Branch point A
1. snRNA forms base-pairs with 5´ end of intron, and at branch site.
Spliceosome
A
5´
3´
2. snRNPs associate with other factors to form spliceosome.
Lariat
A
5´
3´
3. 5´ end of intron is removed and forms bond at branch site,
forming a lariat. The 3´ end of the intron is then cut.
Exon 1
5´
Excised
intron
Exon 2
Mature mRNA
3´
4. Exons are joined; spliceosome disassembles.
37
Alternative splicing
• Single primary transcript can be spliced into
different mRNAs by the inclusion of different sets
of exons
• 15% of known human genetic disorders are due
to altered splicing
• 35 to 59% of human genes exhibit some form of
alternative splicing
• Explains how 25,000 genes of the human
genome can encode the more than 80,000
different mRNAs
38
5’ ut
exon 1
DNA
intron 1
exon 2
intron 2
3’ ut
exon 3
hnRNA
RNA splicing
mRNA 1
RNA splicing
translation
mRNA 2
translation
protein 1
Differential RNA splicing
can lead to different protein products
protein 2
tRNA and Ribosomes
• tRNA molecules carry amino acids to the
ribosome for incorporation into a
polypeptide
– Aminoacyl-tRNA synthetases add amino
acids to the acceptor stem of tRNA
– Anticodon loop contains 3 nucleotides
complementary to mRNA codons
40
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2D “Cloverleaf” Model
3D Ribbon-like Model
Acceptor end
Acceptor end
3‫׳‬
5‫׳‬
Anticodon
loop
3D Space-filled Model
Acceptor end
Anticodon loop
Anticodon loop
Icon
Acceptor end
Anticodon end
c: Created by John Beaver using ProteinWorkshop, a product of the RCSB PDB, and built using the Molecular Biology Toolkit developed by
John Moreland and Apostol Gramada (mbt.sdsc.edu). The MBT is fi nanced by grant GM63208
41
tRNA charging reaction
• Each aminoacyl-tRNA synthetase
recognizes only 1 amino acid but several
tRNAs
• Charged tRNA – has an amino acid added
using the energy from ATP
– Can undergo peptide bond formation without
additional energy
• Ribosomes do not verify amino acid
attached to tRNA
42
43
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Amino
group
NH3+
ATP
Pi Pi
Carboxyl
group
Trp
C
O
O–
Amino
acid site
tRNA
site
Aminoacyl-tRNA
synthetase
44
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Amino
group
NH3+
ATP
Pi Pi
tRNA
site
Carboxyl
group
Trp
C
O
O–
Amino
acid site
Accepting
site
OH
tRNA
Aminoacyl-tRNA
Anticodon
synthetase
specific to tryptophan
45
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Amino
group
NH3+
ATP
Pi Pi
tRNA
site
Carboxyl
group
Trp
C
Charged tRNA travels to ribosome
O
O–
Amino
acid site
NH3+
Trp
Accepting
site
C
O
O
OH
tRNA
Aminoacyl-tRNA
Anticodon
synthetase
specific to tryptophan
Charged
tRNA
dissociates
46
• The ribosome has multiple tRNA binding
sites
– P site – binds the tRNA attached to the
growing peptide chain
– A site – binds the tRNA carrying the
next amino acid
– E site – binds the tRNA that carried the
last amino acid
47
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Large
subunit
3´
Small
subunit
Large
subunit
90°
Small
subunit
Large
subunit
0°
mRNA
Small
subunit
5´
48
• The ribosome has two primary functions
– Decode the mRNA
– Form peptide bonds
• Peptidyl transferase
– Enzymatic component of the ribosome
– Forms peptide bonds between amino acids
49
Translation
• In prokaryotes, initiation complex includes
– Initiator tRNA charged with N-formylmethionine
– Small ribosomal subunit
– mRNA strand
• Ribosome binding sequence (RBS) of
mRNA positions small subunit correctly
• Large subunit now added
• Initiator tRNA bound to P site with A site
empty
50
51
• Initiations in eukaryotes similar except
– Initiating amino acid is methionine
– More complicated initiation complex
– Lack of an RBS – small subunit binds to 5′
cap of mRNA
52
• Elongation adds amino acids
– 2nd charged tRNA can bind to empty A
site
– Requires elongation factor called EF-Tu
to bind to tRNA and GTP
– Peptide bond can then form
– Addition of successive amino acids
occurs as a cycle
53
54
55
• There are fewer tRNAs than codons
• Wobble pairing allows less stringent
pairing between the 3′ base of the codon
and the 5′ base of the anticodon
• This allows fewer tRNAs to accommodate
all codons
56
• Termination
– Elongation continues until the ribosome
encounters a stop codon
– Stop codons are recognized by release
factors which release the polypeptide from the
ribosome
57
58
Protein targeting
• In eukaryotes, translation may occur in the
cytoplasm or the rough endoplasmic reticulum
(RER)
• Signal sequences at the beginning of the
polypeptide sequence bind to the signal
recognition particle (SRP)
• The signal sequence and SRP are recognized
by RER receptor proteins
• Docking holds ribosome to RER
• Beginning of the protein-trafficking pathway
59
60
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RNA
RNA polymerase
polymerase IIII
1. RNA polymerase
II in the nucleus
copies one
strand
of the DNA to
produce the
primary
transcript.
3´
5´
Primary
Primary RNA
RNA transcript
transcript
61
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
RNA
RNA polymerase
polymerase IIII
1. RNA polymerase
II in the nucleus
copies one
strand
of the DNA to
produce the
primary
transcript.
3´
Primary
Primary RNA
RNA transcript
transcript
5´
Primary
Primary RNA
RNA transcript
transcript
2. The primary transcript
is processed by
addition of a 5´
methyl-G cap,
cleavage and
polyadenylation of the
3´ end, and removal of
introns. The mature
mRNA is then
exported through
nuclear pores to the
cytoplasm.
Poly-A tail
tail
Poly-A
Cut intron
intron
Cut
Mature
Mature mRNA
mRNA
5´ cap
cap
5´
62
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
RNA
RNA polymerase
polymerase IIII
1. RNA polymerase
II in the nucleus
copies one
strand
of the DNA to
produce the
primary
transcript.
3´
Primary RNA transcript
5´
Primary
Primary RNA
RNA transcript
transcript
2. The primary transcript
is processed by
addition of a 5´
methyl-G cap,
cleavage and
polyadenylation of the
3´ end, and removal of
introns. The mature
mRNA is then
exported through
nuclear pores to the
cytoplasm.
Poly-A tail
Cut intron
Mature mRNA
5´ cap
3. The 5´ cap of the
mRNA
associates with
the small subunit
of the ribosome. 5´ cap
The initiator
tRNA and large
subunit are
added to form
an initiation
complex.
Large
subunit
mRNA
Small
subunit
Cytoplasm
63
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
RNA
RNA polymerase
polymerase IIII
1. RNA polymerase
II in the nucleus
copies one
strand
of the DNA to
produce the
primary
transcript.
3´
5´
Primary
Primary RNA
RNA transcript
transcript
2. The primary transcript
is processed by
addition of a 5´
methyl-G cap,
cleavage and
polyadenylation of the
3´ end, and removal of
introns. The mature
mRNA is then
exported through
nuclear pores to the
cytoplasm.
Primary RNA transcript
Poly-A tail
Cut intron
3. The 5´ cap of the
mRNA
associates with
the small subunit
of the ribosome.
The initiator
tRNA and large
subunit are
added to form
an initiation
complex.
Mature mRNA
5´ cap
Large
subunit
5´ cap
mRNA
Small
subunit
Cytoplasm
Cytoplasm
Amino acids
tRNA arrivesin A site
3´
mRNA
5´
A site
P site
E site
4. The ribosome cycle begins with the
growing peptide attached to the tRNA
in the P site. The next charged tRNA
binds to the A site with its anticodon
complementary to the codon in the
mRNA in this site.
64
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
RNA
RNA polymerase
polymerase IIII
1. RNA polymerase
II in the nucleus
copies one
strand
of the DNA to
produce the
primary
transcript.
3´
5´
Primary
Primary RNA
RNA transcript
transcript
2. The primary transcript
is processed by
addition of a 5´
methyl-G cap,
cleavage and
polyadenylation of the
3´ end, and removal of
introns. The mature
mRNA is then
exported through
nuclear pores to the
cytoplasm.
Primary RNA transcript
Poly-A tail
Cut intron
3. The 5´ cap of the
mRNA
associates with
the small subunit
of the ribosome.
The initiator
tRNA and large
subunit are
added to form
an initiation
complex.
Cytoplasm
Amino acids
tRNA arrivesin A site
3´
Mature mRNA
5´ cap
Large
subunit
5´ cap
mRNA
Small
subunit
Cytoplasm
Lengthening
polypeptide chain
Emptyt
RNA
3´
mRNA
5´
A site
P site
E site
4. The ribosome cycle begins with the
growing peptide attached to the tRNA
in the P site. The next charged tRNA
binds to the A site with its anticodon
complementary to the codon in the
mRNA in this site.
5´
5. Peptide bonds form between the
amino terminus of the next amino
acid and the carboxyl terminus of
the growing peptide. This transfers
the growing peptide to the tRNA in
the A site, leaving the tRNA in the
P site empty.
65
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
RNA
RNA polymerase
polymerase IIII
1. RNA polymerase
II in the nucleus
copies one
strand
of the DNA to
produce the
primary
transcript.
3´
5´
Primary
Primary RNA
RNA transcript
transcript
2. The primary transcript
is processed by
addition of a 5´
methyl-G cap,
cleavage and
polyadenylation of the
3´ end, and removal of
introns. The mature
mRNA is then
exported through
nuclear pores to the
cytoplasm.
Primary RNA transcript
Poly-A tail
Cut intron
5´ cap
3. The 5´ cap of the
mRNA
associates with
the small subunit
of the ribosome.
The initiator
tRNA and large
subunit are
added to form
an initiation
complex.
Cytoplasm
Amino acids
tRNA arrivesin A site
3´
Large
subunit
5´ cap
mRNA
Small
subunit
Cytoplasm
Empty tRNA moves into
E site and is ejected
Lengthening
polypeptide chain
Emptyt
RNA
Mature mRNA
3´
3´
mRNA
5´
A site
P site
E site
4. The ribosome cycle begins with the
growing peptide attached to the tRNA
in the P site. The next charged tRNA
binds to the A site with its anticodon
complementary to the codon in the
mRNA in this site.
5´
5. Peptide bonds form between the
amino terminus of the next amino
acid and the carboxyl terminus of
the growing peptide. This transfers
the growing peptide to the tRNA in
the A site, leaving the tRNA in the
P site empty.
5´
6. Ribosome translocation moves the
ribosome relative to the mRNA and
its bound tRNAs. This moves the
growing chain into the P site, leaving
the empty tRNA in the E site and the
A site ready to bind the next
charged tRNA.
66
67
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68
Mutation: Altered Genes
• Point mutations alter a single base
• Base substitution – substitute one base for
another
– Silent mutation – same amino acid inserted
– Missense mutation – changes amino acid
inserted
• Transitions
• Transversions
– Nonsense mutations – changed to stop codon
69
70
71
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Normal
Deoxygenated
Tetramer
Normal HBB Sequence
Polar
Leu
C
T
Thr
G
A
C
Pro
T
C
C
Glu
T
G
A
Glu
G
A
A
Lys
G
A
A
Ser
G
T
C
Amino acids
T Nucleotides
Abnormal
Deoxygenated
Tetramer
1
2
1 2
1
2
1 2
Hemoglobin
tetramer
"Sticky" nonpolar sites
Abormal HBB Sequence
Nonpolar (hydrophobic)
Leu
C
T
Thr
G
A
C
val
Pro
T
C
C
T
G
T
Glu
G
A
A
Lys
G
A
A
Ser
G
T
C
Amino acids
T Nucleotides
Tetramers form long chains
when deoxygenated. This
distorts the normal red blood
cell shape into a sickle shape.
72
• Frameshift mutations
– Addition or deletion of a single base
– Much more profound consequences
– Alter reading frame downstream
– Triplet repeat expansion mutation
• Huntington disease
• Repeat unit is expanded in the disease allele
relative to the normal
73
Chromosomal mutations
• Change the structure of a chromosome
– Deletions – part of chromosome is lost
– Duplication – part of chromosome is copied
– Inversion – part of chromosome in reverse
order
– Translocation – part of chromosome is moved
to a new location
74
75
76
• Mutations are the starting point for
evolution
• Too much change, however, is harmful to
the individual with a greatly altered
genome
• Balance must exist between amount of
new variation and health of species
77