Download Chapter 10 - Protein Synthesis: Transcription and Translation

Protein Synthesis:
Transcription and Translation
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From nucleus to cytoplasmaa
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transcription
DNA
mRNA
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protein
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translation
trait
nucleus
cytoplasm
Review
Central Dogma of Molecular Biology
DNA vs RNA
Sugar:
Bonds with Adenine:
# of Strands:
DNA
RNA
deoxyribose
ribose
thymine
uracil
two
one
Kinds of RNA
• Messenger RNA (mRNA):copies DNA’s
code & carries the genetic information to
the ribosomes
• Ribosomal RNA (rRNA):along with
protein, makes up the ribosomes
• Transfer RNA (tRNA): transfers amino
acids to the ribosomes where proteins are
synthesized
Transcription
• Making mRNA from DNA
• ONE DNA strand is the
template (pattern)
– match bases
• A: U
• G:C
• Enzyme
– RNA polymerase
Sense vs. Antisense Strand
• The antisense strand is the template
strand during transcription, which is copied
for translation into a protein.
• a sense strand, or coding strand, is the
strand of DNA running from 5' to 3' that is
complementary to the antisense strand of
DNA, which runs from 3' to 5'.
• The sense strand is the strand of DNA that
has the same sequence as the mRNA.
DNA contains codes
Transcription in prokaryotes
• RNA polymerase recognizes a specific
base sequence in the DNA called a
promoter and binds to it.
• The promoter identifies the start of a gene,
which strand is to be copied, and the
direction that it is to be copied.
RNA POLYMERASE- adds RNA nucleotides
complimentary to the DNA template strand
• Complimentary bases are assembled (U
instead of T).
• A termination code in the DNA indicates
where transcription will stop in
prokaryotes.
• The mRNA produced is called a mRNA
transcript.
Transcription Eukaryotes
• Step 1: Transcription factors proteins
bind and double stranded DNA unzips
as RNA polymerase binds to promoter
(TATAA sequence “TATA box” or other
promoter)
T
Matching bases of DNA & RNA
• Step 2: Match RNA bases
to DNA bases on one of the
DNA strands. Built 5’ 3’,
using DNA 3’-5’ template
A
G
U
A
G
G
U
U
C
A
AG
C
C
G
A
U
A
C
A C C
RNA
polymerase
A
U
G
T G G T A C A G C T A G T C A T CG T A C CG T
U
C
Matching bases of DNA & RNA
• U instead of T is matched to A and
the mRNA transcript is built 5’-3’
DNA
mRNA
TACGCACA TTTA CGTA CG
AUGCGUGUAAAUGCAUGC
Ending Transcription
• Step 3:
Transcription
stops upon
reading
polyadenylation
signal
sequence.
RNA Polymerase
5’ 3’
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mRNA Processing in
Eukaryotes
• After the DNA is transcribed
into RNA, editing must be done
to the nucleotide chain to make
the RNA functional
• Introns, non-functional
segments of DNA are snipped
out of the chain by snRNP’s
(spliceosomes or ribonucleases)
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mRNA Editing
• Exons, segments of DNA that
code for proteins, are then
rejoined by the enzyme ligase
• A guanine triphosphate cap is
added to the 5” end of the
newly copied mRNA
• A poly A tail is added to the 3’
end of the RNA
• The newly processed mRNA can
then leave the nucleus through 17
nuclear pores
Result of Transcription
CAP
New Transcript
Tail
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mRNA Transcript
•mRNA leaves the nucleus
through its pores and goes to
the ribosomes
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How does mRNA code for
proteins
• mRNA leaves nucleus
• mRNA goes to ribosomes in cytoplasm
• Proteins built from instructions on mRNA
mRNA
A C C A U G U C G A U C A GU A GC A U G GC A
How?
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Translation
mRNA to protein = Translation
• The working instructions  mRNA
• The reader  ribosome
• The transporter  transfer RNA (tRNA)
ribosome
mRNA
A C C A U G U C G A U C A GU A GC A U G GC A
U GG
tRNA
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U A C
tRNA
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A G
tRNA
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C
U AG
tRNA
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Translation
• Translation is the process of
decoding the mRNA into a
polypeptide chain
• Ribosomes read mRNA three
bases or 1 codon at a time and
construct the proteins
• The process begins by the mRNA
leaving the nucleus through
nuclear pores
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Messenger RNA (mRNA)
• Carries the information for a
specific protein
• Made up of 500 to 1000
nucleotides long
• Sequence of 3 bases called
codon
• AUG – methionine or start
codon
• UAA, UAG, or UGA – stop
codons
Initiation and Termination Codes
• An initiation code “start codon” signals
the start of a genetic message. As the
ribosome moves along a mRNA transcript,
it will not begin synthesizing protein until it
reaches an initiation code. (Ex. AUG)
• Termination codes “stop codon” signal
the end of the genetic message. Synthesis
stops when the ribosome reaches a
terminator codon.(Ex. UAA, UAG, UGA)
mRNA codes for proteins in
triplets
DNA
TACGCACATTTACGTACGCGG
codon
mRNA
AUGCGUGUAAAUGCAUGC
AUGCGUGUAAAUGCAUGCGCC
Met
protein
Cys Ala
Arg
ribosome
?
Val Asn Ala
 Codon = block of 3 mRNA bases
The Genetic Code
•Use the code by
reading from the
center to the outside
•Example: AUG codes
for Methionine
•Genetic code
contains a wobble at
the third b.p.-there is
redundancy in the
genetic code, resulting
in fewer errors.
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Genetic Code- Another way
Name the Amino Acids
•
•
•
•
•
GGG?
UCA?
CAU?
GCA?
AAA?
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Translation
overview
Ribosomes
• Made of a large and small subunit
• Composed of rRNA (40%) and
proteins (60%)
• Have three sites for tRNA
attachment --- A –accepting site
and P site- peptide bond
• The tRNA exits from the E site
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Transfer RNA (tRNA)
A
C
• Clover-leaf shape
3’
C
5’
• Single stranded molecule
with attachment site at 3’
end for an amino acid
• Opposite end has three
nucleotide bases called the
anticodon
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Transfer RNA
The amino acid is bound to the 3’
end by a covalent ester bond
through a condensation reaction
This is done by aminoacyl-tRNA
synthetases or tRNA activating
enzyme- one for every a.a.
tRNA is held together by
intramolecular base pairing
There is a specific tRNA molecule
for every codon or for every amino
acid
Codons and Anticodons
• Recall a codon is made from
3 bases within mRNA
• The 3 bases of an anticodon
are complimentary to the 3
bases of a codon
• Example: Codon ACU
Anticodon UGA
UGA
ACU
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The Process
Step 1- Initiation
• mRNA transcript start
codon AUG attaches to
the small ribosomal
subunit
• Small subunit attaches
to large ribosomal
subunit
mRNA transcript
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tRNA Binding Steps
1. The amino acid will react with ATP and
become activated (with aid of enzyme
ATPase). The ATP lose energy in this
process
2. The activated amino acid will then bind to
the acceptor stem (3’) of its own tRNA by a
covalent bond aided by tRNA synthetase
– The bond between the tRNA and the a.a is broken when the
peptide bond is formed
Step 2 - Elongation
• As the ribosome moves, one tRNA carrying the Met
amino acid moves into the A site, where the anticodon
matches the mRNA transcript (AUG).
• It then moves to the P site of the ribosome and another
tRNA brings the next amino acid
• Peptide bonds join the amino acids and leaves the P site
tRNA empty (done by peptidyl transferase)
• The empty tRNA is moved to the E site where it exits
Ribosome
“walks” the
strand of
mRNA 3 bases
at a time 5’-3’
1. Transfer of proper tRNA to
A-site of ribosome.
2. peptidyl
transfer/
formation of
peptide bond
3. translocation
Step 3-Termination
• When the ribosome reads a stop codon.
• When this code (UGA, UAA, UAG) is
reached protein translation is terminated.
• Release factor binds
• Polypeptide dissociates from ribosome
• Occurs once per protein
End Product –The Protein!
• The end products of protein
synthesis is a primary structure
of a protein
• A sequence of amino acid
bonded together by peptide
bonds
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aa1
aa3
aa4
aa5
aa199
aa200
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Ribosomes
Large
subunit
E
Site
P
Site
A
Site
mRNA
Small subunit
A U G
C U
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Initiation
aa2
aa1
2-tRNA
G A U
anticodon
1-tRNA
U A C
hydrogen
bonds
A U G
codon
C U A C
mRNA
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Elongation
peptide bond
aa1
aa3
aa2
3-tRNA
1-tRNA
anticodon
hydrogen
bonds
U A C
A U G
codon
2-tRNA
G A A
G A U
C U A C U U C G A
mRNA
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aa1
peptide bond
aa3
aa2
1-tRNA
3-tRNA
U A C
(leaves)
2-tRNA
A U G
G A A
G A U
C U A C U U C G A
mRNA
Ribosomes move over one codon
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aa1
peptide bonds
aa4
aa2
aa3
4-tRNA
2-tRNA
A U G
3-tRNA
G C U
G A U G A A
C U A C U U C G A A C U
mRNA
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aa1
peptide bonds
aa4
aa2
aa3
2-tRNA
4-tRNA
G A U
(leaves)
3-tRNA
A U G
G C U
G A A
C U A C U U C G A A C U
mRNA
Ribosomes move over one codon
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aa1
peptide bonds
aa5
aa2
aa3
aa4
5-tRNA
U G A
3-tRNA
4-tRNA
G A A G C U
G C U A C U U C G A A C U
mRNA
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peptide bonds
aa1
aa5
aa2
aa3
aa4
5-tRNA
U G A
3-tRNA
G A A
4-tRNA
G C U
G C U A C U U C G A A C U
mRNA
Ribosomes move over one codon
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aa4
aa5
Termination
aa199
aa3 primary
structure
aa2 of a protein
aa200
aa1
200-tRNA
A C U
terminator
or stop
codon
C A U G U U U A G
mRNA
http://www.youtube.com/watch?v=5iS4CR
PPDus
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Mutation
• A mutation is a change in amino acid
sequence and can be either a;
• Frameshift (deletion)
Original DNA: THE BIG RED ANT ATE ONE FAT BUG
Frameshift mutation: THB IGR EDA NTA TEO NEF ATB UG?
• Point Mutation
Original DNA: THE BIG RED ANT ATE ONE FAT BUG
Point mutation: THA BIG RED ANT ATE ONE FAT BUG
Silent, Missense, and Nonsense
Mutations
• Three kinds of point mutations can occur.
A mutation that results in an amino acid
substitution is called a missense mutation.
• A mutation that results in a stop codon so
that incomplete proteins are produced, it is
called a nonsense mutation.
• A mutation that produces a functioning
protein is called a silent mutation.
Terms
• Splicesomes• removes introns and ligase joins
• RNA polymerase• adds RNA nucleotides complimentary to the DNA template strand
• Transcription factors• proteins that recognize specific sequences in DNA when making mRNA
and help RNA polymerase bind
• ATPase• converts ATP to ADP and releases energy to do work ( used to bond
tRNA to mRNA and GTPase also used when adding a.a to tRNA)
• tRNA synthetase (tRNA activating enzyme) • enzyme that catalyzes the esterification of a specific amino acid to a
specific tRNA
• Peptidyltransferase• forms peptide bonds and helps with protein release
• recognizes tRNA code and adds specific a.a. (a covalent bond)
• Protein release factors• Helps release peptide during termination of translation by recognizing
the termination codon or stop codon in an mRNA sequence.
Question:
 What would be the
complimentary RNA strand
for the following DNA
sequence?
DNA 5’-GCGTATG-3’
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Answer:
• DNA 5’-GCGTATG-3’
• RNA 3’-CGCAUAC-5’
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Exercise - Understanding DNA,
mRNA, tRNA, and protein.
Template (anti-sense)
strand
Non-template strand
GGG
TAC
mRNA
tRNA
Amino Acid
CUU
UCG
Leu
CCU