IX: DNA Function: Protein Synthesis

1
IX DNA Function Protein Synthesis A.
Overview B. Transcription C. RNA
Processing D. Deciphering the Genetic Code
2
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)
3
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 non-overlapping.
4
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.
5
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-RNAs, ribosomes, and amino
acids, the chemical reactions would make protein
based on this m-RNA sequence. (in vitro)
polypeptide
6
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-RNAs, 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 AAs had been incorporated, and at what
fractions.
60
40
7
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-RNAs, 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 AAs had been incorporated, and at what
fractions. Homopolymers were easy make
UUUUUUU RNA, get polypeptide with only
phenylalanine
8
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-RNAs, 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 AAs 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).
9
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)
10
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
11
(No Transcript)
12
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!!
13
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.
5
3
M-RNA
C G C A U A C A C A A
U G U
5
3
14
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. 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.
15
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 T A T A C G T A C A A A C G G T T A T A C T A
C T T T
T G A T A T G C A T G T T T G C C A A T A T G A T
G A A A
nonsense
3
5
intron
exon
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!
16
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 anti-parallel
nature of the double helix, and the chemical
limitations of the 'reading' enzyme, RNA
Polymerase.
Promoter
3
5
sense
A C T A T A C G T A C A A A C G G T T A T A C T A
C T T T
T G A T A T G C A T G T T T G C C A A T A T G A T
G A A A
nonsense
3
5
intron
exon
exon
Promoters have sequences recognized by the RNA
Polymerase. They bind in particular orientation.
17
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 anti-parallel
nature of the double helix, and the chemical
limitations of the 'reading' enzyme, RNA
Polymerase.
Promoter
3
5
sense
A C T A T A C G T A C A A A C G G T T A T A C T A
C T T T
G C A U GUUU G C C A A U AUG A U G A
T G A T A T G C A T G T T T G C C A A T A T G A T
G A A A
nonsense
3
5
intron
exon
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).

18
VI. Protein Synthesis A. Overview B. The Process
of Protein Synthesis 1. Transcription c.
Transcription ends at a sequence called the
'terminator'.
Promoter
Terminator
3
5
sense
A C T A T A C G T A C A A A C G G T T A T A C T A
C T T T
G C A U GUUU G C C A A U AUG A U G A
T G A T A T G C A T G T T T G C C A A T A T G A T
G A A A
nonsense
3
5
intron
exon
exon
Terminator sequences destabilize the RNA
Polymerase and the enzyme decouples from the DNA,
ending transcription
19
VI. Protein Synthesis A. Overview B. The Process
of Protein Synthesis 1. Transcription c.
Transcription ends at a sequence called the
'terminator'.
Promoter
Terminator
3
5
sense
A C T A T A C G T A C A A A C G G T T A T A C T A
C T T T
G C A U GUUU G C C A A U AUG A U G A
T G A T A T G C A T G T T T G C C A A T A T G A T
G A A A
nonsense
3
5
intron
exon
exon
Initial RNA PRODUCT
20
VI. Protein Synthesis A. Overview B. The Process
of Protein Synthesis 1. Transcription c.
Transcription ends at a sequence called the
'terminator'.
Promoter
Terminator
3
5
sense
A C T A T A C G T A C A A A C G G T T A T A C T A
C T T T
T G A T A T G C A T G T T T G C C A A T A T G A T
G A A A
nonsense
3
5
21
VI. Protein Synthesis A. Overview B. The Process
of Protein Synthesis 1. Transcription 2.
Transcript Processing
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.
22
VI. Protein Synthesis A. Overview B. The Process
of Protein Synthesis 1. Transcription 2.
Transcript Processing
AUG A
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 m-RNA and a recipe for a
protein.
23
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)
24
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)
25
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.
26
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)
27
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
28
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).
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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 AAs
30
E. Translation!!! 1. Players a.
processed m-RNA transcript b. Ribosome
c. T-RNA and AAs d. Protein
factors increase efficiency of process
31
E. Translation!!! 1. Players 2. Process
a. Charging t-RNAs
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.
32
E. Translation!!! 1. Players 2. Process
a. Charging t-RNAs b. Initiation -
METH-t-RNA binds to SRS in p-site, forming the
Initiation Complex
33

• E. Translation!!!
• 1. Players
• 2. Process
• a. Charging t-RNAs
• 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

34
E. Translation!!! 1. Players 2. Process
a. Charging t-RNAs b. Initiation
c. Elongation (Polymerization) -The second
AA-t-RNA complex binds in the Acyl site.
35
E. Translation!!! 1. Players 2. Process
a. Charging t-RNAs 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 AAs.
36
E. Translation!!! 1. Players 2. Process
a. Charging t-RNAs 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 AAs. - Uncharged t-RNA shifts to
e-site And is released from ribosome, while the
m-RNA, t-RNA complex shifts to the p-site
37
E. Translation!!! 1. Players 2. Process
a. Charging t-RNAs 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 AAs. - Uncharged t-RNA shifts to
e-site And is released from ribosome, while the
m-RNA, 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
38
E. Translation!!! 1. Players 2. Process
a. Charging t-RNAs 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
39

• E. Translation!!!
• 1. Players
• 2. Process
• a. Charging t-RNAs
• b. Initiation
• c. Elongation (Polymerization)
• And another translocation reaction occurs

40

• E. Translation!!!
• 1. Players
• 2. Process
• a. Charging t-RNAs
• b. Initiation
• c. Elongation (Polymerization)
• And another translocation reaction occurs. This
  is repeated until.

41
E. Translation!!! 1. Players 2. Process
a. Charging t-RNAs 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.
42
E. Translation!!! 1. Players 2. Process 3.
Polysomes
M-RNAs last for only minutes or hours before
their bases are cleaved and recycled.
Productivity is amplified by having multiple
ribosomes reading down the same m-RNA molecule
creating the polysome structure seen here.
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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 C A U G U U U G C C A A U
U G A
45
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 C A U G U U U G C C A A U
U G A
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.
46
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.
M-RNA
G C A U G U U U G C C A A U
U G A
47
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
M-RNA
G C A U G U U U G C C A A U
U G A
48
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
G C A U G U U U G C C A A U
U G A
The amino acids are bound and the ribosome moves
3-bases downstream
49
VI. Protein Synthesis A. Overview B. The Process
of Protein Synthesis 1. Transcription 2.
Transcript Processing 3. Translation e.
polymerization proceeds
Meth
Phe
M-RNA
G C A U G U U U G C C A A U
U G A
The amino acids are bound and the ribosome moves
3-bases downstream
50
VI. Protein Synthesis A. Overview B. The Process
of Protein Synthesis 1. Transcription 2.
Transcript Processing 3. Translation e.
polymerization proceeds
Meth
Phe
Ala
M-RNA
G C A U G U U U G C C A A U
U G A
The amino acids are bound and the ribosome moves
3-bases downstream
51
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
M-RNA
G C A U G U U U G C C A A U
U G A
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.
52
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.
53
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.
54
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.
55
IX DNA Function Protein Synthesis A.
Overview 1. The central dogma of genetics
unidirectional flow of information 2. The
code is - linear
56
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 AAs found in living
systems.
57
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.
58
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.
59
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.
60
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.
61
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
62
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.