Transcript Camp 1 - UCSC Directory of individual web sites

Chapter 25
Gene Expression and Protein Synthesis
Introduction
• The central dogma of molecular biology
• Information contained in DNA molecules is expressed
in the structure of proteins.
• Gene expression is the turning on or activation of a
gene.
DNA
replication
DNA
RNA
replication
Transcription mRNA
Re vers e tran s criptase
eukaryotes (humans)
In Nucleus of cell
Translation
protein
Eukaryotes (humans)
In cytoplasm
Bacteria (transcriptn + translatn)
Transcription
•
-the process by which information encoded in a DNA molecule is
copied into an mRNA molecule.
• 1. Transcription starts when the DNA double helix begins to unwind near
the gene to be transcribed.
• 2. Only one strand of the DNA is transcribed.
• 3. Ribonucleotides assemble along the unwound DNA strand in a
complementary sequence.
• Enzymes called polymerases (poly) catalyze transcription:
•
poly I: formation of  rRNA formation,
•
poly II: formation of  mRNA formation
•
poly III: formation of  tRNA formation.
1.
2.
3.
4.
Transcription
•
A eukaryotic gene has two RNA; the parts:
1.
2.
3.
4.
5.
A structural gene that is transcribed into structural gene is made of
exons and introns.
A regulatory gene that controls transcription; the regulatory gene is not
transcribed but has control elements, one of which is the promoter.
A promoter is unique to each gene.
There is always a sequence of bases on the DNA strand called an
initiation signal.
Promoters also contain consensus sequences, such as the TATA box, in
which the two nucleotides T and A are repeated many times.
3.
5. 4.
Figure 25.2
2.
1.
RNA in Translation
•
•
•
•
mRNA, rRNA, and tRNA all participate in translation.
Protein synthesis takes place on ribosomes (e.g. rRNA).
A ribosome dissociates into larger (60S) and a smaller body (40S).
The 5’ end of the mature mRNA is bonded to the 40S ribosome and
this unit then joined to the 60S ribosome.
• Triplets of bases on mRNA are called codons. e.g. AUG
• The 20 amino acids are then brought to the mRNA-ribosome
complex, each amino acid by its own particular tRNA (e.g. w/ Met).
rRNA-40S
mRNA
rRNA-60S
Note: anticodon for AUG:
UAC  “Met” see table 25.1
tRNA
tRNA
•
•
•
Each tRNA is specific for only one amino acid (e.g. UAC  Met).
Cell carries at least 20 specific enzymes (e.g. AARS) each specific for one amino acid.
(i.e. links Amino acids with spec. tRNA)
•
NOTE: 20 amino acids always/usually present in cytoplasm to make proteins (usually)
otherwise-bad hair day? Poor nutrition. . . Limited amino acid(s) in diet
•
•
Challenge Question? What is the only food with all 20 amino acids in one serving?
•
Most important segments of tRNA
• 1. site where enzymes attach amino acids (3’ end)
• 2. recognition site. (three basses anticodon: UAC
bondS AUG)
(e.g. Met)
1.
Expanded tRNA
(e.g. Met)
(AARS)
2. anticodon
UAC
mRNA
tRNA
Confirming your Knowledge
If a codon is CGU (mRNA) what is it’s anticodon?
What amino acid does the code for? Hint see table 25.1
mRNA
The Genetic Code
• Assignments of triplets is based on several types
of experiments.
• One of these used synthetic mRNA.
• If mRNA is polyU, polyPhe is formed; the triplet UUU,
therefore, must code for Phe.
• If mRNA is poly ---ACACAC---, poly(Thr-His) is formed;
ACA must code for Thr, and CAC for His.
• By 1967, the genetic code was broken.
The Genetic Code: Table 25.1
5'
U
C
U
UUU
UUC
UUA
UUG
CUU
CUC
CUA
CUG
AU U
A AU C
AU A
AU G
GU U
G GU C
GU A
GU G
Phe
Phe
Leu
Leu
Leu
Leu
Leu
Leu
Ile
Ile
Ile
Met*
Val
Val
Val
Val
C
UCU
UCC
UCA
UCG
Ser
Ser
Ser
Ser
A
U AU
U AC
U AA
U AG
CAU
CAC
CAA
CAG
Tyr
Tyr
Stop
Stop
His
His
Gln
Gln
G
U GU
U GC
U GA
U GG
CGU
CGC
CGA
CGG
Cys
Cys
S top
Trp
Arg
Arg
Arg
Arg
CCU
CCC
CCA
CCG
Pro
Pro
Pro
Pro
ACU
ACC
ACA
ACG
GCU
GCC
GCA
GCG
Thr
Thr
Thr
Thr
Ala
Ala
Ala
Ala
AAU
AAC
AAA
AAG
GAU
GAC
GAA
GAG
As n
As n
Lys
Lys
A sp
A sp
Glu
Glu
A GU
A GC
A GA
A GG
GGU
GGC
GGA
GGG
Ser
Ser
Arg
Arg
Gly
Gly
Gly
Gly
3'
U
C
A
G
U
C
A
G
U
C
A
G
U
C
A
G
*AUG s ign als tran slation initiation as w ell as codin g for Met
NOTE:
Features of the Code
• All 64 codons have been assigned.
• 61 code for amino acids. (What about the others 3?)
• 3 (UAA, UAG, and UGA) serve as
termination signals.
• AUG, universal start signal.
• Only Trp and Met have one codon each.
• More than one triplet can code for the same amino acid; Leu, Ser,
and Arg, for example, are each coded for by six triplets.
5'
U
AUG CGUAGUAAUGGUAGUAUAUAA
Start codon
Stop codon
C
U
UUU
UUC
UUA
UUG
CUU
CUC
CUA
CUG
AU U
A AU C
AU A
AU G
GU U
G GU C
GU A
GU G
Phe
Phe
Leu
Leu
Leu
Leu
Leu
Leu
Ile
Ile
Ile
Met*
Val
Val
Val
Val
C
UCU
UCC
UCA
UCG
Ser
Ser
Ser
Ser
A
U AU
U AC
U AA
U AG
CAU
CAC
CAA
CAG
Tyr
Tyr
Stop
Stop
His
His
Gln
Gln
G
U GU
U GC
U GA
U GG
CGU
CGC
CGA
CGG
Cys
Cys
S top
Trp
Arg
Arg
Arg
Arg
CCU
CCC
CCA
CCG
Pro
Pro
Pro
Pro
ACU
ACC
ACA
ACG
GCU
GCC
GCA
GCG
Thr
Thr
Thr
Thr
Ala
Ala
Ala
Ala
AAU
AAC
AAA
AAG
GAU
GAC
GAA
GAG
As n
As n
Lys
Lys
A sp
A sp
Glu
Glu
A GU
A GC
A GA
A GG
GGU
GGC
GGA
GGG
Ser
Ser
Arg
Arg
Gly
Gly
Gly
Gly
3'
U
C
A
G
U
C
A
G
U
C
A
G
U
C
A
G
*AUG s ign als tran slation initiation as w ell as codin g for Met
Features of the Code
• For the 15 amino acids coded for by 2, 3, or 4 triplets, it
is only the third letter of the codon that varies. Gly, for
example, is coded for by GGA, GGG, GGC, and GGU.
• The code is almost universal: it the same in viruses,
prokaryotes, and eukaryotes; the only exceptions are
some codons in mitochondria.
• Supports Darwins theory of evolution
Confirming your Knowledge
If you had 750 DNA segment, (assume Met (AUG)
and stop codon UUG
are stripped off )
How many Amino acids appear in the
protein the DNA codes for?
Translation
• the process whereby a base sequence of mRNA
is used to create a protein.
• There are four major stages in protein synthesis:
•
•
•
•
1. Activation
2. Initiation
3. Elongation
4. Termination
DNA
replication
DNA
RNA
replication
Transcription mRNA
Re vers e tran s criptase
Translation
protein
Protein Synthesis
1. Hydrolize ATP AMP
2. Link adenosine to Amino acid
• Activation
1.
2.
Amino Acid Activation
• 1. Activated amino acid is bound to spec. tRNA
• 2. w/ ester carboxyl group of the amino acid and
the 3’-OH of the tRNA.
(e.g. Cys)
2.
1.
(e.g. Cys)
(AARS)
Chain Initiation
•
40S
Figure 25.4 Formation of an
initiation complex.
1.
mRNA
1. 40S rRNA binds with mRNA
40S
3.
2.
2. tRNA binds with 40S rRNA/mRNA
3. 60S rRNA binds with 40S rRNA/mRNA
60S
P site A site
E site
60S
Acceptor (A) Site
Protein (P) Site
Exit (E) Site (tRNA)
Elongation: Figure 25.6
Show Videos
26.6
E site
Process:
Hydrolyzes GTP GDP
A site
P site
Forms peptide bond (b/w Ala-Met)
Peptide Bond Formation
• Peptide bond formation in protein synthesis.
X-ray model of
Ribosome
w/ rRNA
tRNA
mRNA
Fig. 25-7, p. 629
UAA (terminate me (‘.’)
Termination
• Chain termination requires:
• Termination codons (UAA, UAG, or UGA) of mRNA.
• Releasing factors that cleave the polypeptide chain
from the last tRNA and release the tRNA from the
ribosome.
Gene Regulation
• the various methods used by organisms to
control which genes will be expressed and when.
• 1. Regulations operate at the transcriptional level (DNA  RNA)
• 2. Others operate at the translational level (mRNA  protein).
1. Transcriptional Level
In eukaryotes, transcription regulated by 3 elements:
promoters,
enhancers,
and response elements.
2. Translational Level
• a number of mechanisms that ensure quality
control.
• A. aminoacyl-tRNA synthase (AARS) control
Each amino acid must bond to the proper tRNA.
B. Termination control
stop codons must be recognized by release factors.
C. Post-translational control
• In most proteins, the Met at the N-terminal end is
removed by Met-aminopeptidase.
• Certain proteins called chaperones help newly
synthesized proteins to fold properly.
Mutations and Mutagens
• Mutation: a heritable change in the base
sequence of DNA.
• It is estimated that, on average, there is one copying
error for every 1010 bases.
• Mutations can occur during replication.
• Base errors can also occur during transcription in
protein synthesis (a nonheritable error).
• Other errors in replication may lead to a change in
protein structure and be very harmful.
Mutations and Mutagens
•
•
Chemical(s) that causes a base change in DNA.
What are common mutagens we are all exposed to?
•
Cells have repair mechanisms called
nucleotide excision repair
(NER) units  prevents mutations.
• NER can prevent mutations by cutting out damaged areas and
resynthesizing them.
• Not all mutations are harmful.
• Certain ones may be beneficial because they enhance the survival
rate of the species.
Recombinant DNA
• DNA from two sources that have been combined into one
molecule.
• One example of the technique begins with plasmids found
in the cells of Escherichia coli.
• plasmid: a small, circular, double-stranded
DNA molecule of bacterial origin.
• A class of enzymes called restriction endonucleases cleave DNA
at specific locations.
• One, for example, may be specific for cleavage of the bond
between A-G in the sequence -CTTAAAG-.
Restriction enzyme cleaves here . . .
Recombinant DNA
• In this example “B ” stands for bacterial gene, and “H
Sticky ends
for human gene.
B
B
GAATTC
CTTAAG
restriction
B en don ucleas e
B
B
B
G
+ AATTC
CTTAA
G
B
B
• The DNA is now double-stranded with two “sticky
ends”, each with free bases that can pair with a
complementary section of DNA.
• Next, we cut a human gene with the same restriction
endonuclease; for example, the gene for human
insulin.
H
H
GAATTC
CTTAAG
restriction
H en don ucleas e
H
H
H
G
+ AATTC
CTTAA
G
H
H
Recombinant DNA
• The human gene is now spliced into the plasmid by the
enzyme DNA ligase.
B
B
G
AATTC
+
CTTAA
G
H D N A ligase
H
B
B
GAATTC
CTTAAG
H
H
• Splicing takes place at both ends of the human gene and the
plasmid is once again circular.
• The modified plasmid is then put back into the bacterial cell where
it replicates naturally every time the cell divides.
• These cells now manufacture the human protein, in our example
human insulin, by transcription and translation.
Recombinant DNA
• Figure 25.11 The recombinant
DNA technique.
1st human insulin produced
by fermentation
1972
In 1972, University of California, San Francisco, biochemist Herbert Boyer met
Stanford University geneticist Stanley Norman Cohen. Cohen founded Genentech 1976
Cloning DNA
• Figure 26.17 The
cloning of human
DNA fragments
with a viral vector.
Gene Therapy
• Figure 26.18 Gene therapy
via retroviruses.
Protein Synthesis
Gene Expression and
Protein Synthesis
End
Chapter 26