chapter 8a Gene Expression 2.pdf

8/9/2019 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.

8-6


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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

10


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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

12


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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

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chapter 8a Gene Expression 2.pdf