IX: DNA Function: Protein Synthesis

1
IX DNA Function Protein Synthesis
2
IX DNA Function Protein Synthesis A.
Overview 1. The central dogma of genetics
unidirectional flow of information
3
IX DNA Function Protein Synthesis A.
Overview 1. The central dogma of genetics
unidirectional flow of information 2. Why a
two-step process? a. Historical
contingency Thats how it evolved from an RNA?
Protein system
4
IX DNA Function Protein Synthesis A.
Overview 1. The central dogma of genetics
unidirectional flow of information 2. Why a
two-step process? - it evolved that way. -
because it is more productive.
tRNA
5
IX DNA Function Protein Synthesis A.
Overview 1. The central dogma of genetics
unidirectional flow of information 2. The
code is 3. Why a two-step process? 4.
Players ds-DNA GENE (recipe) RNA
polymerases make m-RNA (gene
transcript) r-RNA (reader in ribosome) t-RNA
(AA carrier)
tRNA
6
IX DNA Function Protein Synthesis A.
Overview B. Transcription
7
IX DNA Function Protein Synthesis A.
Overview B. Transcription 1. The DNA
template In a given region (gene), only one
strand is transcribed only one strand carries a
message that makes sense. The sequence on the
other strand is limited to being complementary to
the first strand.
3
5
sense strand
C A T
3
5
G T A
anti-sense strand
8
IX DNA Function Protein Synthesis A.
Overview B. Transcription 1. The DNA
template In a given region (gene), only one
strand is transcribed only one strand carries a
message that makes sense. The sequence on the
other strand is limited to being complementary to
the first strand. There is a region upstream
from the gene called the PROMOTER. This is where
the RNA Polymerase binds. The polymerase is
attracted to particular sequences. Many are
consensus sequences found upstream from different
genes, and across many many species.
3
5
sense strand
C A T
3
5
G T A
anti-sense strand
9
IX DNA Function Protein Synthesis A.
Overview B. Transcription 1. The DNA
template In a given region (gene), only one
strand is transcribed only one strand carries a
message that makes sense. The sequence on the
other strand is limited to being complementary to
the first strand. There is a region upstream
from the gene called the PROMOTER. This is where
the RNA Polymerase binds. The polymerase is
attracted to particular sequences. Many are
consensus sequences found upstream from different
genes, and across many many species. In all
bacteria, the sequence TATAAT lies 10 bases
upstream from all bacterial genes, and TTGACA
lies 35 bases upstream. Two binding sites create
a directionality. Promoters can be 40 bases
long. Frequency of binding is affected by
variation in the rest of the sequence.
3
5
sense strand
C A T
3
5
G T A
anti-sense strand
10
IX DNA Function Protein Synthesis A.
Overview B. Transcription 1. The DNA
template In a given region (gene), only one
strand is transcribed only one strand carries a
message that makes sense. The sequence on the
other strand is limited to being complementary to
the first strand. In all eukaryotes, there is
a consensus sequence of TATA in all promoters at
-35, and a CAAT box at -80. There are also
enhancer regions that can modulate binding, and
Transcription Factors that bind to the promoter
and increase/decrease the efficacy of polymerase
binding.
3
5
sense strand
C A T
3
5
G T A
anti-sense strand
11
IX DNA Function Protein Synthesis A.
Overview B. Transcription 1. The DNA
template In a given region (gene), only one
strand is transcribed only one strand carries a
message that makes sense. The sequence on the
other strand is limited to being complementary to
the first strand. In all eukaryotes, there is
a consensus sequence of TATA in all promoters at
-35, and a CAAT box at -80. There are also
enhancer regions that can modulate binding, and
Transcription Factors that bind to the promoter
and increase/decrease the efficacy of polymerase
binding. Why is there consensus in promoter
sequences across all life? What does that say
about how well mutations are tolerated in these
regions?
3
5
sense strand
C A T
3
5
G T A
anti-sense strand
12
IX DNA Function Protein Synthesis A.
Overview B. Transcription 1. The DNA
template In a given region (gene), only one
strand is transcribed only one strand carries a
message that makes sense. The sequence on the
other strand is limited to being complementary to
the first strand. There is region downstream
called a TERMINATOR (40 bases long).
3
5
sense strand
C A T
3
5
G T A
anti-sense strand
13
IX DNA Function Protein Synthesis A.
Overview B. Transcription 1. The DNA
template In a given region (gene), only one
strand is transcribed only one strand carries a
message that makes sense. The sequence on the
other strand is limited to being complementary to
the first strand. So, the polymerase binds at
the promoter, transcribes the whole gene, and
decouples at the terminator.
3
5
sense strand
C A T
3
5
G T A
anti-sense strand
14
IX DNA Function Protein Synthesis A.
Overview B. Transcription 1. The DNA
template In a given region (gene), only one
strand is transcribed only one strand carries a
message that makes sense. The sequence on the
other strand is limited to being complementary to
the first strand. In bacteria, the gene is a
continuous coding sequence.
3
5
sense strand
Continuous recipe for a protein
3
5
anti-sense strand
15
IX DNA Function Protein Synthesis A.
Overview B. Transcription 1. The DNA
template In a given region (gene), only one
strand is transcribed only one strand carries a
message that makes sense. The sequence on the
other strand is limited to being complementary to
the first strand. In bacteria, the gene is a
continuous coding sequence. In eukaryotes,
genes contain non-coding, intervening sequences
called introns the coding sequences are called
exons.
3
5
sense strand
Disconti
nuous recipe for
a protein
3
5
anti-sense strand
16
IX DNA Function Protein Synthesis A.
Overview B. Transcription 1. The DNA
template In a given region (gene), only one
strand is transcribed only one strand carries a
message that makes sense. The sequence on the
other strand is limited to being complementary to
the first strand. In bacteria, the gene is a
continuous coding sequence. In eukaryotes,
genes contain non-coding, intervening sequences
called introns the coding sequences are called
exons. Although transcription is continuous and
every base is transcribed, RNA processing is
required to splice out the non-coding introns and
create a continuous reading frame for
translation.
3
5
sense strand
Disconti
nuous recipe for
a protein
3
5
anti-sense strand
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TANGENT
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There are two alternate hypotheses for the
evolution of introns
Archaea (introns in r-RNA and t-RNA genes)
Eubacteria (no introns at all)
Eukarya (introns)
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There are two alternate hypotheses for the
evolution of introns Introns Early The
ancestral structure was a split gene structure,
favored because evolution could proceed rapidly
by the shuffling of functional exons to create
new genes (exon shuffling hypothesis).
Archaea (introns in r-RNA and t-RNA genes)
Eubacteria (no introns at all)
Eukarya (introns)
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There are two alternate hypotheses for the
evolution of introns Introns Early The
ancestral structure was a split gene structure,
favored because evolution could proceed rapidly
by the shuffling of functional exons to create
new genes (exon shuffling hypothesis).
Archaea (introns in r-RNA and t-RNA genes)
Eubacteria (no introns at all)
Eukarya (introns)
Introns were lost from prokaryotes because of the
extreme selective advantage for rapid division.
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There are two alternate hypotheses for the
evolution of introns Introns Late The
ancestral structure was a continuous gene
structure. Introns evolved as transposeable
elements and inserted themselves and multiplied
only in eukaryotic ancestors.
Archaea (introns in r-RNA and t-RNA genes)
Eubacteria (no introns at all)
Eukarya (introns)
22
The split-gene structure of eukaryotes was
discovered in 1977 Philip Sharp and coworkers
found that viral genes in eukaryotes and initial
transcripts were longer than the functional m-RNA
or proteins.
23
The split-gene structure of eukaryotes was
discovered in 1977 Philip Sharp and coworkers
found that viral genes in eukaryotes and initial
transcripts were longer than the functional m-RNA
or proteins. 1978 Walter Gilbert coins the
terms intron and exon.
24
Heteroduplex analyses of DNA and m-RNA show
loops of RNA that have no complement in the DNA
template strand
25
All eukaryotic genes except those coding for
histones have introns some make up the vast
majority of the gene, itself
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IX DNA Function Protein Synthesis A.
Overview B. Transcription 1. The DNA
template 2. RNA Polymerase In bacteria,
there is only one enzyme consisting of a core
enzyme (responsible for polymerization), and
subunits that affect different functions. For
example, the sigma subunit is responsible for
initiation, and the rho subunit stimulates
termination.
3
5
sense strand
C A T
3
5
G T A
anti-sense strand
27
IX DNA Function Protein Synthesis A.
Overview B. Transcription 1. The DNA
template 2. RNA Polymerase In bacteria,
there is only one enzyme consisting of a core
enzyme (responsible for polymerization) with two
polypeptides, and subunits that affect different
functions. For example, the sigma subunit is
responsible for initiation, and the rho subunit
stimulates termination. There are different
sigma subunits that affect polymerase binding
complementing the variations in promoters.
3
5
sense strand
C A T
3
5
G T A
anti-sense strand
28
IX DNA Function Protein Synthesis A.
Overview B. Transcription 1. The DNA
template 2. RNA Polymerase In bacteria,
there is only one enzyme consisting of a core
enzyme (responsible for polymerization) with two
polypeptides, and subunits that affect different
functions In eukaryotes, there are three RNA
Polymerases
3
5
sense strand
C A T
3
5
G T A
anti-sense strand
29
IX DNA Function Protein Synthesis A.
Overview B. Transcription 1. The DNA
template 2. RNA Polymerase 3. RNA
triphosphate precursors
3
5
sense strand
C A T
3
5
G T A
anti-sense strand
A
U
A
U
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IX DNA Function Protein Synthesis A.
Overview B. Transcription 1. The DNA
template 2. RNA Polymerase 3. RNA
triphosphate precursors 4. Process in
Bacteria
3
5
sense strand
C A T
3
5
G T A
anti-sense strand
31
IX DNA Function Protein Synthesis A.
Overview B. Transcription 1. The DNA
template 2. RNA Polymerase 3. RNA
triphosphate precursors 4. Process in
Bacteria a. Polymerase (with sigma) lands on
ds-DNA at promoter
3
5
sense strand
C A T
3
5
G T A
anti-sense strand
32
IX DNA Function Protein Synthesis A.
Overview B. Transcription 1. The DNA
template 2. RNA Polymerase 3. RNA
triphosphate precursors 4. Process in
Bacteria a. Polymerase (with sigma) lands on
ds-DNA at promoter b. Helicases separate
strands first bases linked 5 ? 3
3
5
sense strand
C C C A G T C A T G G G T.
G G G
3
5
3
anti-sense strand
5
33
IX DNA Function Protein Synthesis A.
Overview B. Transcription 1. The DNA
template 2. RNA Polymerase 3. RNA
triphosphate precursors 4. Process in
Bacteria a. Polymerase (with sigma) lands on
ds-DNA at promoter b. Helicases separate
strands first bases linked 5 ? 3 c. Sigma
subunit dissociates, polymerization
continues
3
5
sense strand
C C C A G T C A T G G G T.
G G G UC A GU A C C C A
3
5
3
anti-sense strand
5
34
IX DNA Function Protein Synthesis A.
Overview B. Transcription 1. The DNA
template 2. RNA Polymerase 3. RNA
triphosphate precursors 4. Process in
Bacteria a. Polymerase (with sigma) lands on
ds-DNA at promoter b. Helicases separate
strands first bases linked 5 ? 3 c. Sigma
subunit dissociates, polymerization
continues d. Termination sequences rich
in Cs and Gs, followed by As and Ts
3
5
sense strand
C C C C G C A A G C G G G G A A T T.
G G G GC G UU C G C C C C U U A A
3
5
3
35
IX DNA Function Protein Synthesis A.
Overview B. Transcription 1. The DNA
template 2. RNA Polymerase 3. RNA
triphosphate precursors 4. Process in
Bacteria a. Polymerase (with sigma) lands on
ds-DNA at promoter b. Helicases separate
strands first bases linked 5 ? 3 c. Sigma
subunit dissociates, polymerization
continues d. Termination sequences rich
in Cs and Gs, followed by As and Ts -
Rho Independent - the Cs and Gs in the
m-RNA for a stem-loop structure and
binds to a protein bound to the Polymerase
(nusA)
3
5
sense strand
C C C C G C A A G C G G G G A A T T.
3
5
3
U C G C C C C
G G G GC G U
36
IX DNA Function Protein Synthesis A.
Overview B. Transcription 1. The DNA
template 2. RNA Polymerase 3. RNA
triphosphate precursors 4. Process in
Bacteria a. Polymerase (with sigma) lands on
ds-DNA at promoter b. Helicases separate
strands first bases linked 5 ? 3 c. Sigma
subunit dissociates, polymerization
continues d. Termination sequences rich
in Cs and Gs, followed by As and Ts -
Rho Independent - the Cs and Gs in the
m-RNA for a stem-loop structure and
binds to a protein bound to the Polymerase
(nusA) - this causes the polymerase to pause,
just as it is reading the area rich in As which
have fewer h-bonds. The pausing and the
destabilization of the polymerase caused by the
stem-loop causes the m-RNA/polymerase to detach.
3
5
sense strand
C C C C G C A A G C G G G G A A T T.
3
5
3
U C G C C C C
G G G GC G U
37
(No Transcript)
38
IX DNA Function Protein Synthesis A.
Overview B. Transcription 1. The DNA
template 2. RNA Polymerase 3. RNA
triphosphate precursors 4. Process in
Bacteria a. Polymerase (with sigma) lands on
ds-DNA at promoter b. Helicases separate
strands first bases linked 5 ? 3 c. Sigma
subunit dissociates, polymerization
continues d. Termination sequences rich
in Cs and Gs, followed by As and Ts -
Rho Independent - recent research has
identified certain RNAs that loop and bind a
small protein produced BY their own code. So, if
the product concentration is high, the m-RNA
binds the protein, loops, and shuts down
transcription (down-regulating the gene). These
RNAs that turn off their own gene are
riboswitches
3
5
sense strand
C C C C G C A A G C G G G G A A T T.
3
5
3
U C G C C C C
G G G GC G U
39
IX DNA Function Protein Synthesis A.
Overview B. Transcription 1. The DNA
template 2. RNA Polymerase 3. RNA
triphosphate precursors 4. Process in
Bacteria a. Polymerase (with sigma) lands on
ds-DNA at promoter b. Helicases separate
strands first bases linked 5 ? 3 c. Sigma
subunit dissociates, polymerization
continues d. Termination sequences rich
in Cs and Gs, followed by As and Ts -
Rho-dependent Rho a circular hexamer, binds
the m-RNA in C-G rich regions
3
5
sense strand
C C C C G C A A G C G G G G A A T T.
G G G GC G UU C G C C C C U U A A
3
5
3
40
IX DNA Function Protein Synthesis A.
Overview B. Transcription 1. The DNA
template 2. RNA Polymerase 3. RNA
triphosphate precursors 4. Process in
Bacteria a. Polymerase (with sigma) lands on
ds-DNA at promoter b. Helicases separate
strands first bases linked 5 ? 3 c. Sigma
subunit dissociates, polymerization
continues d. Termination sequences rich
in Cs and Gs, followed by As and Ts -
Rho-dependent Rho a circular hexamer, binds
the m-RNA in C-G rich regions it slides up the
strand decoupling the polymerase from the
m-RNA at A-T rich sites.
3
5
sense strand
C C C C G C A A G C G G G G A A T T.
G G G GC G UU C G C C C C U U A A
3
5
3
41
IX DNA Function Protein Synthesis A.
Overview B. Transcription 1. The DNA
template 2. RNA Polymerase 3. RNA
triphosphate precursors 4. Process in
Bacteria a. Polymerase (with sigma) lands on
ds-DNA at promoter b. Helicases separate
strands first bases linked 5 ? 3 c. Sigma
subunit dissociates, polymerization
continues d. Termination e. Polycistronic
DNA In bacteria, proteins involved in the
same metabolic process are often encoded by
neighboring genes that are read as a UNIT
(operon),
Three genes read as a single unit
42
IX DNA Function Protein Synthesis A.
Overview B. Transcription 1. The DNA
template 2. RNA Polymerase 3. RNA
triphosphate precursors 4. Process in
Bacteria a. Polymerase (with sigma) lands on
ds-DNA at promoter b. Helicases separate
strands first bases linked 5 ? 3 c. Sigma
subunit dissociates, polymerization
continues d. Termination e. Polycistronic
DNA In bacteria, proteins involved in the
same metabolic process are often encoded by
neighboring genes that are read as a UNIT
(operon), producing one m-RNA that has the
transcript of all three genes
Three genes read as a single unit
43
IX DNA Function Protein Synthesis A.
Overview B. Transcription 1. The DNA
template 2. RNA Polymerase 3. RNA
triphosphate precursors 4. Process in
Bacteria a. Polymerase (with sigma) lands on
ds-DNA at promoter b. Helicases separate
strands first bases linked 5 ? 3 c. Sigma
subunit dissociates, polymerization
continues d. Termination e. Polycistronic
DNA In bacteria, proteins involved in the
same metabolic process are often encoded by
neighboring genes that are read as a UNIT
(operon), producing one m-RNA that has the
transcript of all three genes then, in
TRANSLATION, ribosomes attach at start codons
along the strand, synthesizing all proteins
simultaneously.
Three genes read as a single unit
44
IX DNA Function Protein Synthesis A.
Overview B. Transcription 1. The DNA
template 2. RNA Polymerase 3. RNA
triphosphate precursors 4. Process in
Bacteria Translation of m-RNA by ribosomes
occurs even before m-RNA is complete!
45
IX DNA Function Protein Synthesis A.
Overview B. Transcription 1. The DNA
template 2. RNA Polymerase 3. RNA
triphosphate precursors 4. Process in
Bacteria 5. Process in Eukaryotes
46
IX DNA Function Protein Synthesis A.
Overview B. Transcription 1. The DNA
template 2. RNA Polymerase 3. RNA
triphosphate precursors 4. Process in
Bacteria 5. Process in Eukaryotes - The
chromatin must be unwound chromatin
remodeling
47
IX DNA Function Protein Synthesis A.
Overview B. Transcription 1. The DNA
template 2. RNA Polymerase 3. RNA
triphosphate precursors 4. Process in
Bacteria 5. Process in Eukaryotes - The
chromatin must be unwound chromatin
remodeling - Initiation is regulated by
enhancer sequences upstream and downstream from
the gene, and transcription factor binding.
48
IX DNA Function Protein Synthesis A.
Overview B. Transcription 1. The DNA
template 2. RNA Polymerase 3. RNA
triphosphate precursors 4. Process in
Bacteria 5. Process in Eukaryotes - The
chromatin must be unwound chromatin
remodeling - Initiation is regulated by
enhancer sequences upstream and downstream from
the gene, and transcription factor binding. The
enhancer sequences are cis-acting regulatory
elements (CREs), because they are on the same
chromosome as the gene. Since the transcription
factors are proteins encoded elsewhere in the
genome, even on different chromosomes, they are
called trans-acting regulatory elements.
49
IX DNA Function Protein Synthesis A.
Overview B. Transcription 1. The DNA
template 2. RNA Polymerase 3. RNA
triphosphate precursors 4. Process in
Bacteria 5. Process in Eukaryotes - The
chromatin must be unwound chromatin
remodeling - Initiation is regulated by
enhancer sequences upstream and downstream from
the gene, and transcription factor binding. -
Because the m-RNA is bound in the
nucleusseparated from the ribosomestranslation
does not take place immediately.
50
IX DNA Function Protein Synthesis A.
Overview B. Transcription 1. The DNA
template 2. RNA Polymerase 3. RNA
triphosphate precursors 4. Process in
Bacteria 5. Process in Eukaryotes - The
chromatin must be unwound chromatin
remodeling - Initiation is regulated by
enhancer sequences upstream and downstream from
the gene, and transcription factor binding. -
Because the m-RNA is bound in the
nucleusseparated from the ribosomestranslation
does not take place immediately. - Most
significantly, the initial RNA product is
PROCESSED.
51
IX DNA Function Protein Synthesis A.
Overview B. Transcription C. RNA
Processing
52
IX DNA Function Protein Synthesis A.
Overview B. Transcription C. RNA
Processing - In prokaryotes, only r-RNA genes
have introns protein-encoding genes have a
continuous coding sequence.
53
IX DNA Function Protein Synthesis A.
Overview B. Transcription C. RNA
Processing - In prokaryotes, only r-RNA genes
have introns protein-encoding genes have a
continuous coding sequence. As such, the m-RNA
can be translated as soon as it is made.
54
IX DNA Function Protein Synthesis A.
Overview B. Transcription C. RNA
Processing - In eukaryotes, the initial RNA
transcripts contain introns that are spliced
out.
55
IX DNA Function Protein Synthesis A.
Overview B. Transcription C. RNA
Processing - In eukaryotes, the initial RNA
transcripts contain introns that are spliced
out. - In addition a 7-mG cap and poly-A tail
are added to the processed RNA probably to
reduce the rate of exonuclease activity in the
cytoplasm.
56
IX DNA Function Protein Synthesis A.
Overview B. Transcription C. RNA
Processing - In eukaryotes, the initial RNA
transcripts contain introns that are spliced
out. - In addition a 7-mG cap and poly-A tail
are added to the processed RNA probably to
reduce the rate of exonuclease activity in the
cytoplasm). - Group I introns splice
themselves out of r-RNA they have auto-catalytic
function. These were the first RNA molecules
found that had enzyme-like catalytic properties
ribozymes (Cech, 1982)
Free guanine nucleoside
57
IX DNA Function Protein Synthesis A.
Overview B. Transcription C. RNA
Processing - Group I introns splice
themselves out of r-RNA they have auto-catalytic
function. These were the first RNA molecules
found that had enzyme-like catalytic properties
ribozymes (Cech, 1982) - Group II introns are
autocatalytic, too, and occur in mitochondria and
chloroplast m,t-RNA.
58
IX DNA Function Protein Synthesis A.
Overview B. Transcription C. RNA
Processing - Group I introns - Group II
introns - nuclear introns m-RNA transcripts
in the nucleus are very large, and processing is
more complicated
59
IX DNA Function Protein Synthesis A.
Overview B. Transcription C. RNA
Processing - Group I introns - Group II
introns - nuclear introns m-RNA transcripts
in the nucleus are very large, and processing is
more complicated. Terminal GU-AG sequences are
recognized by snRPs (snurps) that have
sn-RNAs rich in Uracil (U designations). sn-RN
A short, nuclear RNA also U-RNA snRPs
short, nuclear, riboproteins
60
IX DNA Function Protein Synthesis A.
Overview B. Transcription C. RNA
Processing - Group I introns - Group II
introns - nuclear introns m-RNA transcripts
in the nucleus are very large, and processing is
more complicated. Terminal GU-AG sequences are
recognized by snRNPs (snurps) that have
sn-RNAs rich in Uracil (U designations).
Binding of complementary snRPs creates the
spliceosome, which creates a lariat structure in
the RNA.
61
IX DNA Function Protein Synthesis A.
Overview B. Transcription C. RNA
Processing - Group I introns - Group II
introns - nuclear introns m-RNA transcripts
in the nucleus are very large, and processing is
more complicated. Terminal GU-AG sequences are
recognized by snRNPs (snurps) that have
sn-RNAs rich in Uracil (U designations).
Binding of complementary snRPs creates the
spliceosome, which creates a lariat structure in
the RNA. The intron is cleaved, and the exons
are ligated together.
62
IX DNA Function Protein Synthesis A.
Overview B. Transcription C. RNA
Processing - Group I introns - Group II
introns - nuclear introns m-RNA transcripts
in the nucleus are very large, and processing is
more complicated. Terminal GU-AG sequences are
recognized by snRNPs (snurps) that have
sn-RNAs rich in Uracil (U designations).
Binding of complementary snRPSs creates the
spliceosome, which creates a lariat structure in
the RNA. The intron is cleaved, and the exons
are ligated together. This splicing can vary,
such that a single m-RNA can be spliced at
different places and produce different
proteinsultimately from the same gene!
63
IX DNA Function Protein Synthesis A.
Overview B. Transcription C. RNA
Processing So, the final product has a 7mG cap,
poly-A tail, and a continuous message. This
mature m-RNA leaves the nucleus and enters the
cytoplasm, where it will be translated.