chapter 8a Gene Expression 2.pdf
Transcript of chapter 8a Gene Expression 2.pdf
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CHAPTER 8 GENE
EXPRESSION
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Geneexpression
Other
RNAs
mRNA coded by ribosome--> translation
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Prokaryotes
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!"#$ &
'('"')"'*( *+ ",)(!-,'$"'*(Figure 8.11
Prokaryotic
signma protein recognize gene & bring pol, where transcription starts & goes to right D
- RNA pol
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!"#$ .#/*(0)"'*(
Prokaryotic
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Double stranded DNA helix
mRNA
TTTACCATGTTTAAAGGGCCCGACTACTTAACT|||||||||||||||||||||||||||||||||AAATGGTACAAATTTCCCGGGCAGATGAATTGA
TAAG-G-G-C-C-C-G-A-C
ATT G-G-G-C-C-C-G-A-C 3'
| | | | | | | | |C-C-C-G-G-G-C-T-G
UA
A
5'UUUACCAUGUUUAAAGGGCCCGUCUACUUAACU
A
G
TC GACTTAGCGTGCATAGCA
||||||||||||||||||CTGAATCGCACGTATCGA
5'
3'
3'
5'
This diagram is full of very important information. It shows a mRNA
molecule being transcribed from a double stranded DNA molecule. The
transcription machinery is not shown.
Notice that:
1. The DNA template strand is being read in the 3' to 5' direction.
2. The RNA molecule is being synthesized in the 5' to 3' direction.
3. The mRNA is antiparallel and complementary to the DNA strand that is
being read.4. About 12 nucleotides of the mRNA are H-bonded to the DNA strand.
Actually, it is just such H-bonding that enables the transcription machinery to
choose the correct ribonucleotides when synthesizing an RNA molecule.
Ribonucleotides are chosen based on their ability to H-bond or base pair to the
DNA template.
transcription 5'-3', add ribosome nucleotides from 5' to 3', melt DNA in front while travelingcom
complementary, antiparallel
elongation
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!"#$ 1
"#,2'()"'*(
Prokaryotic
Helicase breaks H bond
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TranslationDouble stranded DNA helix
mRNA
TTTACCATGTTTAAAGGGCCCGACTACTTAACT|||||||||||||||||||||||||||||||||AAATGGTACAAATTTCCCGGGCAGATGAATTGA
TAAG-G-G-C-C-C-G-A-C
AT
T G-G-G-C-C-C-G-A-C 3'
| | | | | | | | |C-C-C-G-G-G-C-T-G
UA
A
5'UUUACCAUGUUUAAAGGGCCCGUCUACUU
AACU
A
G
TC GACTTAGCGTGCATAGCA
||||||||||||||||||CTGAATCGCACGTATCGA
5'
3'
3'
5'
M F
K
M
F K P GD
5'
3'
3'
5'
ribonucleotides
5' 3'
5' 3'
mRNA
mRNA
amino acidsTranslation
Transcription
N C
N C
N C
N C
N = amino
C = carboxy
protein
protein
protein
protein
PROKARYOTIC
The process of
reading thegenetic code inthe messengerRNA (mRNA)and using that
information tosynthesize thepolypeptide(protein) that isspecified in themRNA.
! Note thedirection ofinformationflow.
ribosomesnascent
protein
! Notepolarity
Prokaryotic
to make protein
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N
NN
5'3'
Initiation Elongation Termination Initiation Elongation Termination
mRNA region of
the first gene (cistron)mRNA region of
the second gene (cistron)
Translation of Prokaryoticpolycistronic mRNA
N
N
Multiple gene--> 1 message, same protein
Ribosomes fall off & split 2 component parts
R added
Protein added
R assemble
Pro: turn on lac operon for lactose
Euk: every gene has its own transcription, produces its own mRNA
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Genetic code
! The Genetic Code: 61triplet codons represent 20amino acids;
! 3 triplet codons signifystop.
64 codons:
61 codons specific 20 amino acids
3 stop codons
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How is the genetic code read?! "34 567689 :;4 ;4:7 46?749 @8 : ;6A A@
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from Atkinson Molecular Biology course 1993-2009
tRNAstRNA single RNA chain folded into 2d cloverleaf
tRNA: small RNA molecules participate in translation of mRNA
not part of ribosome, have extensive 2nd structure
produced by INTRAmolecular base pairing btw nucleotides
-tRNA contains many highly modified &
unusual nucleotides
- following transcription, tRNA contains only
standard set of 4 nucleotides
- Unusual nucleotides are produced by
post-transcription enzymatic modifications
- tRNA has 4 stems & 3 loops (4th loop is
called Variable Loop) locate btw TUC stem
& anticodon stem
- D loop is named for modified nitrogenous base
found within it, dihydrouracil
- TUC loop is named for 4 invariant nucleotides
found within it (thymidylate, pseudouridylate,cytidylate, guanylate)
Anticodon: bp with codon and covalent attach tRNA,brought along with mRNA to correct amino acid
(decode genetic code)
XCCA- amino acid acceptor site, X-any
nucleotides, C-cytidylate, A-adenylate
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Threonine
Thr
eonine
-tRNA thr docdes one of codon specify Threonine
- Codons for threonine: ACU, ACC, ACA, ACG
- tRNA decodes ACU codon
- 3 nucleotides are complementary (bp H
bond & antiparallel
to codon ACU
tRNA is covalently attached to amino acid
threonine
- ACU: starts from 5' to 3', decoded by anticodon UGA
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-Pro: 30-40 different tRNA
-Euk: 50 different tRNA
- Wobble base pairing: a little bit change
of nucleotides Inosine
- genetic code is degenerated by
synonymous codons
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Inosine
Wobble base pairing: non-standard type of base
pairing that occurs between 3rd nucleotides in CODON
& 1st nucleotides in ANTICODON
--> enable single tRNA to recognize more than 1 codon
for particular amino acid.
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Flip it for illustrative purposes.
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-CGA (3rd position)
-CGG: ex of wobble bp
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tRNA
From this slide I only
want you to be sure to
learn at least three
things.1) that the tRNA carries
an amino acid whose
codon that it decodes.
2) that the amino acid is
covalently attached to
the 3 end of the tRNA.
3) that when a tRNA is
carrying an amino acid,
the tRNA is said to becharged.
- tRNA (charged) carrying amino acid (activated)
is aminoacyl tRNA synthetase; charging tRNA
consume energy in form of ATP. Energy from ATP
is conserved in bond btw amino acid & tRNA
- Last 3 nucleotides on 3' end of tRNA is 5' CCA 3'-
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,@D696I49:;4 56IC>4J
9
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2453:8@9I 6H :?68
'8@?:?68 94
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Initiation
Fig. 8.25 a
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Elongation - Addition of amino acids to C terminus polypeptide- Chaged tRNA ushered into A site by elongation factors
- Elongation 5'- 3' d, left to right- 5' message represent the amino term
- As protein grows, we dont add amino acids but TRANSFER amino acid to protein,
transfer the entire protein to AA (buildiing another building??)
-3' carboxylic term
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Fig. 8.25 c
"4;I@8:?68 6H :?68- Simultaneous synthesis of many copies of polypeptide from single mRNA
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$>4:94 ;4Q@4A :9949 6H I=
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How was the genetic code
cracked?
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Experimental demonstration that a genes nucleotidesequence is collinear the amino acid sequence of the
encoded polypeptide. Charles Yanofsky E. coligenes for a subunit of tryptophan
synthetase.
Identified a huge # of mutations. Trp- mutants in trpA. Fine structure recombination mapping used to determine the
position of the mutations in the gene.
Examined a huge number of recombinants
Sequenced the encoded proteins.
Correlated the position of the mutations within the gene to thechanged amino acids.
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8-7Fig. 8.4
Showed that gene is co-linear with theamino acid sequence of the protein.
XO btw codons
1. He wants to know the distance/how far apart 2 genes are/ same or dif
genes--> measure RF
Genetic map
4. Take product of mutant and
make protein (but not working)
--> purify and sequence it
When sequecing it, find wrong
amino acid (15)--> STOP
1 recombinant has no mutation-> wild type gene,
1 recombinant has mutation
3. He can detect
recombination bc 2 mutants
can cross over and give fully
functional Trp2
.
5. The gene is co-linear the sequence of AA- begin of gene is begin of protein ??
49 Glu--> Val, Gln or Met (change of one codon)
not number, just
names
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) 56768 @9 56IC6947 6H I6;4 46?74E
U@V4;48< C6@8< I=F : 9@8B>4 56768E
'8H4;;47 D45:=94 4:53 C6@8< I=F 684:I@86 :5@7E
8-8
1 recombinant has no mutation-> wild type gene,
1 recombinant has mutation
genetic map
He wants to know the distance/how far apart 2 genes are/ same or dif genes--> measure RF
He can detect recombination bc 2 mutants can cross over and give fully functional Trp Then he takes product of each mutant
genes --> protein --> sequence it
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-;@5W :87 X;4884; C;6C694
68B :87 F A@
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Fig. 8.6
Take out 3 nucleotides--> no frameshift
// 2 // --> frameshift
Solve: homologous
recombinaton to put them
together
restore a frameshift -----G---------------
x
----------------A-----
-----G---------A----
insert G, remove A
(b) C f t fl i
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Fig. 8.5b
(b) Consequences of exposure to proflavin
Exposure to proflavin
FC0
rIIB
FC0 FC7
Exposure to proflavin
rIIB
+
revertantOriginalmutation
Secondmutation
riiB+wild-type
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Fig. 8.5c
(c) rIIB+revertant X wild type yields rIIB recombinants.
FC0 FC7
rIIBFC0 rIIB
FC7
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Fig. 8.5d
+
(d) Different sets of mutations generate either a mutant or a normal phenotype.
Proflavin-inducedmutations
( + ) insertion ( ) deletionPhenotype
Mutant
Mutant
or
or
or
or
or
or
or
or+ + + + + +
+ + +
+
+ + + + +
+ + + +
+ +
Mutant
Wild type
Wild type
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...CPhe
(a) Poly- U mRNA encodes polyphenylalanine.
5'
3'
SyntheticmRNA
Analyze radioactivepolypeptides synthesized
Invitro translationalsystem plus radioactiveamino acids
PhePhePhePhePhePheN...
Fig. 8.7
Whatdoeseachcodonspecify?
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[4F 7@956Q4;@49
"34 I,() :87 C;6@84:;E "34 LM 487 6H
I,() 56;;49C6879
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!=II:;F
-6768 5689@9< 6H : 4< 56768 4:53 6H A3@53 9C45@G49 :8 :I@86 :5@7E-674 936A9 : LM :CC@8BE
-674 @85>=749
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Operon
! lacZ, lacY, lacA