IX: DNA Function: Protein Synthesis
A. Overview:
B. Transcription:
C. RNA Processing:
D. Deciphering the Genetic Code
IX: DNA Function: Protein Synthesis
A. Overview:
B. Transcription:
C. RNA Processing:
D. Deciphering the Genetic Code
1. Sidney Brenner – suggested a triplet code
(minimum necessary to encode 20 AA)
IX: DNA Function: Protein Synthesis
A. Overview:
B. Transcription:
C. RNA Processing:
D. Deciphering the Genetic Code
1. Sidney Brenner – suggested a triplet code
(minimum necessary to encode 20 AA)
2. Crick analyzed addition/deletion
mutations, and confirmed a triplet code that is nonoverlapping.
IX: DNA Function: Protein Synthesis
D. Deciphering the Code:
3. Nirenberg and Mattaei – 1961:
Used polynucleotide phosphorylase (enzyme) to create random sequences of RNA
bases – mRNA.
IX: DNA Function: Protein Synthesis
D. Deciphering the Code:
3. Nirenberg and Mattaei – 1961:
Used polynucleotide phosphorylase (enzyme) to create random sequences of RNA
bases – mRNA. Then added t-RNA’s, ribosomes, and amino acids, the chemical
reactions would make protein based on this m-RNA sequence. (in vitro)
polypeptide
IX: DNA Function: Protein Synthesis
D. Deciphering the Code:
3. Nirenberg and Mattaei – 1961:
Used polynucleotide phosphorylase (enzyme) to create random sequences of RNA
bases – mRNA. Then added t-RNA’s, ribosomes, and amino acids, the chemical
reactions would make protein based on this m-RNA sequence. (in vitro) Then they
could isolate and digest the protein and see which AA’s had been incorporated, and at
what fractions….
60%
40%
IX: DNA Function: Protein Synthesis
D. Deciphering the Code:
3. Nirenberg and Mattaei – 1961:
Used polynucleotide phosphorylase (enzyme) to create random sequences of RNA
bases – mRNA. Then added t-RNA’s, ribosomes, and amino acids, the chemical
reactions would make protein based on this m-RNA sequence. (in vitro) Then they
could isolate and digest the protein and see which AA’s had been incorporated, and at
what fractions….
Homopolymers were easy:
make UUUUUUU RNA, get polypeptide with only phenylalanine
IX: DNA Function: Protein Synthesis
D. Deciphering the Code:
3. Nirenberg and Mattaei – 1961:
Used polynucleotide phosphorylase (enzyme) to create random sequences of RNA
bases – mRNA. Then added t-RNA’s, ribosomes, and amino acids, the chemical
reactions would make protein based on this m-RNA sequence. (in vitro) Then they
could isolate and digest the protein and see which AA’s had been incorporated, and at
what fractions….
Homopolymers were easy:
make UUUUUUU RNA, get polypeptide with only phenylalanine
make AAAAAAA RNA, get polypeptide with only lysine
make CCCCCCCC RNA, get polypeptide with only proline
make GGGGGGG RNA, and the molecule folds back on itself… (oh well).
IX: DNA Function: Protein Synthesis
D. Deciphering the Code:
3. Nirenberg and Mattaei – 1961:
Homopolymers were easy:
Heteropolymers were more clever:
add two bases at different ratios (1/6 A, 5/6 C):
IX: DNA Function: Protein Synthesis
D. Deciphering the Code:
3. Nirenberg and Mattaei – 1961:
Homopolymers were easy:
Heteropolymers were more clever:
add two bases at different ratios (1/6 A, 5/6 C):
So, since the enzyme links bases randomly (there is no template), you can
predict how frequent certain 3-base combinations should be:
AAA = 1/6 x 1/6 x 1/6 = 1/216 = 0.4%
IX: DNA Function: Protein Synthesis
D. Deciphering the Code:
3. Nirenberg and Mattaei – 1961: figured out 50 of the 64 codons
4. Khorana - 1962
Dinucleotide, trinucleotides, and tetranucleotides: make specific triplets
He confirmed existing triplets,
filled in others, and identified
stop codons because of
premature termination.
Nobels for Nirenberg and
Khorana!!
IX: DNA Function: Protein Synthesis
D. Deciphering the Code:
5. Patterns
The third position is often
not critical, such that U at
the first position of the
t-RNA (its antiparallel) can
pair with either A or G in
the m-RNA. This reduces
the number of t-RNA
molecules needed.
3’
M-RNA
C G C A U A C A C AA
UGU
5’
3’
5’
IX: DNA Function: Protein Synthesis
D. Deciphering the Code:
5. Patterns
The third position is often
not critical, such that U at
the first position of the tRNA (its antiparallel) can
pair with either A or G in
the m-RNA. This reduces
the number of t-RNA
molecules needed.
There are also some
chemical similarities to the
amino acids encoded by
similar codons, which may
have persisted as the code
evolved because errors
were not as problematic to
protein function.
VI. Protein Synthesis
A. Overview
B. The Process of Protein Synthesis
1. Transcription
a. The message is on one strand of the double helix - the sense strand:
3’
5’
sense
A C TATA C G TA C AAA C G G T TATA C TA C T T T
T GATAT G CAT G T T T G C CAATAT GAT GA A A
5’
nonsense
3’
exon
intron
exon
In all eukaryotic genes and in some prokaryotic
sequences, there are introns and exons. There may
be multiple introns of varying length in a gene. Genes
may be several thousand base-pairs long. This is a
simplified example!
VI. Protein Synthesis
A. Overview
B. The Process of Protein Synthesis
1. Transcription
b. The cell 'reads' the correct strand based on the location of the promoter, the antiparallel nature of the double helix, and the chemical limitations of the 'reading'
enzyme, RNA Polymerase.
3’
Promoter
5’
sense
A C TATA C G TA C AAA C G G T TATA C TA C T T T
T GATAT G CAT G T T T G C CAATAT GAT GA A A
5’
nonsense
3’
exon
intron
exon
Promoters have sequences recognized by the RNA
Polymerase. They bind in particular orientation.
VI. Protein Synthesis
A. Overview
B. The Process of Protein Synthesis
1. Transcription
b. The cell 'reads' the correct strand based on the location of the promoter, the antiparallel nature of the double helix, and the chemical limitations of the 'reading'
enzyme, RNA Polymerase.
3’
Promoter
5’
sense
A C TATA C G TA C AAA C G G T TATA C TA C T T T
G C A U GUUU G C C A A U AUG A U G A
T GATAT G CAT G T T T G C CAATAT GAT GA A A
5’
nonsense
3’
exon
intron
exon
1) Strand separate
2) RNA Polymerase can only synthesize RNA in a 5’3’ direction,
so they only read the anti-parallel, 3’5’ strand (‘sense’ strand).
VI. Protein Synthesis
A. Overview
B. The Process of Protein Synthesis
1. Transcription
c. Transcription ends at a sequence called the 'terminator'.
3’
Promoter
Terminator
5’
sense
A C TATA C G TA C AAA C G G T TATA C TA C T T T
G C A U GUUU G C C A A U AUG A U G A
T GATAT G CAT G T T T G C CAATAT GAT GA A A
5’
nonsense
3’
exon
intron
exon
Terminator sequences destabilize the RNA Polymerase and the
enzyme decouples from the DNA, ending transcription
VI. Protein Synthesis
A. Overview
B. The Process of Protein Synthesis
1. Transcription
c. Transcription ends at a sequence called the 'terminator'.
3’
Promoter
Terminator
5’
sense
A C TATA C G TA C AAA C G G T TATA C TA C T T T
G C A U GUUU G C C A A U AUG A U G A
T GATAT G CAT G T T T G C CAATAT GAT GA A A
5’
3’
exon
Initial RNA
PRODUCT:
nonsense
intron
exon
VI. Protein Synthesis
A. Overview
B. The Process of Protein Synthesis
1. Transcription
c. Transcription ends at a sequence called the 'terminator'.
3’
Promoter
Terminator
5’
sense
A C TATA C G TA C AAA C G G T TATA C TA C T T T
T GATAT G CAT G T T T G C CAATAT GAT GA A A
5’
3’
exon
Initial RNA
PRODUCT:
nonsense
intron
exon
G C A U GUUU G C C A A U AUG A U G A
VI. Protein Synthesis
A. Overview
B. The Process of Protein Synthesis
1. Transcription
2. Transcript Processing
exon
Initial RNA
PRODUCT:
intron
exon
G C A U GUUU G C C A A U AUG A U G A
Introns are spliced out, and exons are spliced together.
Sometimes these reactions are catalyzed by the intron,
itself, or other catalytic RNA molecules called
“ribozymes”.
VI. Protein Synthesis
A. Overview
B. The Process of Protein Synthesis
1. Transcription
2. Transcript Processing
intron
exon
exon
AUG A
Final RNA
PRODUCT:
G C A U GUUU G C C A A U U G A
This final RNA may be complexed with proteins to form
a ribosome (if it is r-RNA), or it may bind amino acids (if
it is t-RNA), or it may be read by a ribosome, if it is mRNA and a recipe for a protein.
IX: DNA Function: Protein Synthesis
A. Overview:
B. Transcription:
C. RNA Processing:
D. Deciphering the Code:
E. Translation!!!
1. Players:
a. processed m-RNA transcript:
binding site (Shine-Delgarno sequence in bacteria: AGGAGG)
(Kazak sequence in eukaryotes: ACCAUGG)
IX: DNA Function: Protein Synthesis
A. Overview:
B. Deciphering the Code:
C. Transcription:
D. RNA Processing:
E. Translation!!!
1. Players:
a. processed m-RNA transcript:
binding site (Shine-Delgarno sequence in bacteria: AGGAGG)
(Kazak sequence in eukaryotes: ACCAUGG)
start codon (AUG)
IX: DNA Function: Protein Synthesis
A. Overview:
B. Deciphering the Code:
C. Transcription:
D. RNA Processing:
E. Translation!!!
1. Players:
a. processed m-RNA transcript:
binding site (Shine-Delgarno sequence in bacteria: AGGAGG)
(Kazak sequence in eukaryotes: ACCAUGG)
start codon (AUG)
codon sequence….
IX: DNA Function: Protein Synthesis
A. Overview:
B. Deciphering the Code:
C. Transcription:
D. RNA Processing:
E. Translation!!!
1. Players:
a. processed m-RNA transcript:
binding site (Shine-Delgarno sequence in bacteria: AGGAGG)
(Kazak sequence in eukaryotes: ACCAUGG)
start codon (AUG)
codon sequence….
stop codon (UGA, etc…)
IX: DNA Function: Protein Synthesis
A. Overview:
B. Deciphering the Code:
C. Transcription:
D. RNA Processing:
E. Translation!!!
1. Players:
a. processed m-RNA transcript:
binding site (Shine-Delgarno sequence in bacteria: AGGAGG)
(Kazak sequence in eukaryotes: ACCAUGG)
start codon (AUG)
codon sequence….
stop codon (UGA, etc…)
7mG cap and poly-A tail in eukaryotes
IX: DNA Function: Protein Synthesis
A. Overview:
B. Deciphering the Code:
C. Transcription:
D. RNA Processing:
E. Translation!!!
1. Players:
a. processed m-RNA transcript:
b. Ribosome:
2 subunits (large and small)
each with a peptidyl site (P)
and aminoacyl site (A).
IX: DNA Function: Protein Synthesis
A. Overview:
B. Deciphering the Code:
C. Transcription:
D. RNA Processing:
E. Translation!!!
1. Players:
a. processed m-RNA transcript:
b. Ribosome
c. T-RNA and AA’s
E. Translation!!!
1. Players:
a. processed m-RNA transcript:
b. Ribosome
c. T-RNA and AA’s
d. Protein factors – increase efficiency of process
E. Translation!!!
1. Players:
2. Process:
a. Charging t-RNA’s
Each t-RNA is bound to a specific
AA by a very specific enzyme; a
unique form of aminoacyl
synthetase.
The specificity of each enzyme is
responsible for the unambiguous
genetic code.
E. Translation!!!
1. Players:
2. Process:
a. Charging t-RNA’s
b. Initiation:
- METH-t-RNA binds to SRS in p-site, forming
the Initiation Complex
E. Translation!!!
1. Players:
2. Process:
a. Charging t-RNA’s
b. Initiation:
-METH-t-RNA binds to SRS in p-site, forming the
Initiation Complex
-The LRS binds to this complex, completing th
aminoacyl site – the first base is in position and
we are ready to polymerize…
E. Translation!!!
1. Players:
2. Process:
a. Charging t-RNA’s
b. Initiation:
c. Elongation (Polymerization):
-The second AA-t-RNA complex binds in the
Acyl site.
E. Translation!!!
1. Players:
2. Process:
a. Charging t-RNA’s
b. Initiation:
c. Elongation (Polymerization):
-The second AA-t-RNA complex binds in the
Acyl site.
-Translocation reaction:
- Peptidyl transferase makes a
Peptide bond between the adjacent AA’s.
E. Translation!!!
1. Players:
2. Process:
a. Charging t-RNA’s
b. Initiation:
c. Elongation (Polymerization):
-The second AA-t-RNA complex binds in the
Acyl site.
-Translocation reaction:
- Peptidyl transferase makes a
Peptide bond between the adjacent AA’s.
- Uncharged t-RNA shifts to e-site
And is released from ribosome, while the mRNA, t-RNA complex shifts to the p-site…
E. Translation!!!
1. Players:
2. Process:
a. Charging t-RNA’s
b. Initiation:
c. Elongation (Polymerization):
-The second AA-t-RNA complex binds in the
Acyl site.
-Translocation reaction:
- Peptidyl transferase makes a
Peptide bond between the adjacent AA’s.
- Uncharged t-RNA shifts to e-site
And is released from ribosome, while the mRNA, t-RNA complex shifts to the p-site…
- the A-site is now open and across
From the next m-RNA codon; ready to accept
The next charged t-RNA
E. Translation!!!
1. Players:
2. Process:
a. Charging t-RNA’s
b. Initiation:
c. Elongation (Polymerization):
-The second AA-t-RNA complex binds in the
Acyl site.
-Translocation reaction
- The third charged t-RNA enters the A-site
E. Translation!!!
1. Players:
2. Process:
a. Charging t-RNA’s
b. Initiation:
c. Elongation (Polymerization):
-And another translocation reaction occurs
E. Translation!!!
1. Players:
2. Process:
a. Charging t-RNA’s
b. Initiation:
c. Elongation (Polymerization):
-And another translocation reaction occurs…. This is repeated until….
E. Translation!!!
1. Players:
2. Process:
a. Charging t-RNA’s
b. Initiation:
c. Elongation (Polymerization):
d. Termination:
When a stop codon is reached (not
the last codon, as shown in the
picture…), no charged t-RNA is
placed in the A-site… this signals
GTP-releasing factors to cleave the
polypeptide from the t-RNA,
releasing it from the ribosome.
E. Translation!!!
1. Players:
2. Process:
3. Polysomes:
M-RNA’s last for only minutes
or hours before their bases are
cleaved and recycled.
Productivity is amplified by
having multiple ribosomes
reading down the same mRNA molecule; creating the
‘polysome’ structure seen
here.
VI. Protein Synthesis
A. Overview
B. The Process of Protein Synthesis
1. Transcription
2. Transcript Processing
3. Translation
a. m-RNA attaches to the ribosome at the 5' end.
M-RNA:
G CAU G U U U G C CAAU U GA
VI. Protein Synthesis
A. Overview
B. The Process of Protein Synthesis
1. Transcription
2. Transcript Processing
3. Translation
a. m-RNA attaches to the ribosome at the 5' end.
M-RNA:
G CAU G U U U G C CAAU U GA
It then reads down the m-RNA, one base at a time, until an ‘AUG’ sequence
(start codon) is positioned in the first reactive site.
VI. Protein Synthesis
A. Overview
B. The Process of Protein Synthesis
1. Transcription
2. Transcript Processing
3. Translation
a. m-RNA attaches to the ribosome at the 5' end.
b. a specific t-RNA molecule, with a complementary UAC anti-codon sequence, binds
to the m-RNA/ribosome complex.
Meth
M-RNA:
G CAU G U U U G C CAAU U GA
VI. Protein Synthesis
A. Overview
B. The Process of Protein Synthesis
1. Transcription
2. Transcript Processing
3. Translation
a. m-RNA attaches to the ribosome at the 5' end.
b. a specific t-RNA molecule, with a complementary UAC anti-codon sequence, binds
to the m-RNA/ribosome complex.
c. A second t-RNA-AA binds to the second site
Phe
Meth
M-RNA:
G CAU G U U U G C CAAU U GA
VI. Protein Synthesis
A. Overview
B. The Process of Protein Synthesis
1. Transcription
2. Transcript Processing
3. Translation
a. m-RNA attaches to the ribosome at the 5' end.
b. a specific t-RNA molecule, with a complementary UAC anti-codon sequence, binds
to the m-RNA/ribosome complex.
c. A second t-RNA-AA binds to the second site
d. Translocation reactions occur
Meth
M-RNA:
Phe
G CAU G U U U G C CAAU U GA
The amino acids are bound and the ribosome moves 3-bases “downstream”
VI. Protein Synthesis
A. Overview
B. The Process of Protein Synthesis
1. Transcription
2. Transcript Processing
3. Translation
e. polymerization proceeds
Meth
M-RNA:
Ala
Asn
Phe
G CAU G U U U G C CAAU U GA
The amino acids are bound and the ribosome moves 3-bases “downstream”
VI. Protein Synthesis
A. Overview
B. The Process of Protein Synthesis
1. Transcription
2. Transcript Processing
3. Translation
e. polymerization proceeds
Meth
M-RNA:
Asn
Phe
Ala
G CAU G U U U G C CAAU U GA
The amino acids are bound and the ribosome moves 3-bases “downstream”
VI. Protein Synthesis
A. Overview
B. The Process of Protein Synthesis
1. Transcription
2. Transcript Processing
3. Translation
e. polymerization proceeds
f. termination of translation
Meth
M-RNA:
Phe
Ala
Asn
G CAU G U U U G C CAAU U GA
Some 3-base codon have no corresponding t-RNA. These are stop codons,
because translocation does not add an amino acid; rather, it ends the chain.
VI. Protein Synthesis
A. Overview
B. The Process of Protein Synthesis
1. Transcription
2. Transcript Processing
3. Translation
4. Post-Translational Modifications
Meth
Phe
Ala
Asn
Most initial proteins need to be modified to be functional. Most need to have
the methionine cleaved off; others have sugar, lipids, nucleic acids, or other
proteins are added.
IX: DNA Function: Protein Synthesis
A. Overview:
B. Deciphering the Code:
C. Transcription:
D. RNA Processing:
E. Translation!!!
- Summary:
The nucleotide sequence in DNA determines the amino acid sequence in proteins. A
single change in that DNA sequence can affect a single amino acid, and may affect the
structure and function of that protein.
IX: DNA Function: Protein Synthesis
A. Overview:
B. Deciphering the Code:
C. Transcription:
D. RNA Processing:
E. Translation!!!
- Summary:
The nucleotide sequence in DNA determines the amino acid sequence in proteins. A
single change in that DNA sequence can affect a single amino acid, and may affect the
structure and function of that protein.
Because all biological processes are catalyzed by either RNA or protienaceous enzymes,
and because proteins are also primary structural, transport, and immunological
molecules in living cells, changes in protein structure can change how living systems
work.
Evolution occurs through changes in DNA, which cause changes in proteins and affect
how and when they act in living cells.
IX: DNA Function: Protein Synthesis
A. Overview:
1. The central dogma of genetics:
unidirectional flow of information
2. The code is:
- linear
IX: DNA Function: Protein Synthesis
A. Overview:
1. The central dogma of genetics:
unidirectional flow of information
2. The code is:
- linear
- ‘triplet’
Three DNA/RNA bases are a ‘word’ that specifies
a single amino acid. This is the minimum
number need to specific the 20 AA’s found in
living systems.
IX: DNA Function: Protein Synthesis
A. Overview:
1. The central dogma of genetics:
unidirectional flow of information
2. The code is:
- linear
- ‘triplet’
- ‘unambiguous’
Each three-base sequence (RNA ‘codon’) codes
for only ONE amino acid.
IX: DNA Function: Protein Synthesis
A. Overview:
1. The central dogma of genetics:
unidirectional flow of information
2. The code is:
- linear
- ‘triplet’
- ‘unambiguous’
- ‘degenerate’ (redundant)
Each amino acid can be coded for by more than
one three-base codon.
IX: DNA Function: Protein Synthesis
A. Overview:
1. The central dogma of genetics:
unidirectional flow of information
2. The code is:
- linear
- ‘triplet’
- ‘unambiguous’
- ‘degenerate’ (redundant)
- ‘start and stop signals’
There are specific codons that signal translation
enzymes where to start and stop.
IX: DNA Function: Protein Synthesis
A. Overview:
1. The central dogma of genetics:
unidirectional flow of information
2. The code is:
- linear
- ‘triplet’
- ‘unambiguous’
- ‘degenerate’ (redundant)
- ‘start and stop signals’
- ‘commaless’
There is no internal punctuation; translation
proceeds from start signal to stop signal.
IX: DNA Function: Protein Synthesis
A. Overview:
1. The central dogma of genetics:
unidirectional flow of information
2. The code is:
- linear
- ‘triplet’
- ‘unambiguous’
- ‘degenerate’ (redundant)
- ‘start and stop signals’
- ‘commaless’
- ‘non-overlapping’
AACGUA is read: ‘AAC’ ‘GUA’
not: ‘AAC’
‘ACG’
‘CGU’
‘GUA’
IX: DNA Function: Protein Synthesis
A. Overview:
1. The central dogma of genetics:
unidirectional flow of information
2. The code is:
- linear
- ‘triplet’
- ‘unambiguous’
- ‘degenerate’ (redundant)
- ‘start and stop signals’
- ‘commaless’
- ‘non-overlapping’
- ‘universal’
With rare exceptions in single codons, all life
forms use the exact same ‘dictionary’… so AAA
codes for lysine in all life. There is one language
of life, suggesting a single origin.