Lecture 11 - prof rami alsaber bio 210 PDF

Preview

Summary

prof rami alsaber bio 210...

Description

Genes and How They Work

1

Prokaryotic transcription • Single RNA polymerase • Initiation of mRNA synthesis does not require a primer • Requires – Promoter – Start site – Termination site

Transcription unit

2

• Promoter – Forms a recognition and binding site for the RNA polymerase – Found upstream of the start site – Not transcribed – Asymmetrical – indicate site of initiation and direction of transcription

3

   ‫׳‬ 

TATAAT– Promoter (–10 sequence) Core enzyme

Holo

Template strand

5′ Downstream 3′

Coding strand

Start site (+1)

TTGACA–Promoter (–35 sequence) Upstream

Prokaryotic RNA polymerase 5′ 3′ b.

a.

 binds to DNA

RNA polymerase bound to unwound DNA

Transcription bubble

5′ 3′

 dissociates ATP Helix opens at –10 sequence Start site RNA synthesis begins 5′ 3′

4

• Elongation – Grows in the 5′-to-3′ direction as ribonucleotides are added – Transcription bubble – contains RNA polymerase, DNA template, and growing RNA transcript – After the transcription bubble passes, the now-transcribed DNA is rewound as it leaves the bubble

5

RNA polymerase DNA Start site

Unwinding

Codingstrand Rewinding

5′

3'

3'

5′ Downstream

3' Upstream

Template strand

mRNA 5′

Transcription bubble

6

• Termination – Marked by sequence that signals “stop” to polymerase • Causes the formation of phosphodiester bonds to cease • RNA–DNA hybrid within the transcription bubble dissociates • RNA polymerase releases the DNA • DNA rewinds

– Hairpin 7

DNA and RNA Polymerase dissociates

RNA polymerase DNA

mRNA dissociates from DNA

3' 5′ 5′ 3'

Four or more U ribonucleotides

mRNA hairpin causes RNA polymerase to pause

Cytosine Guanine Adenine Uracil

5′

8

• Prokaryotic transcription is coupled to translation – mRNA begins to be translated before transcription is finished – Operon • Grouping of functionally related genes • Multiple enzymes for a pathway • Can be regulated together

9

0.25 µm RNA polymerase

mRNA Ribosomes

DNA

Polyribosome Polypeptide chains 10

Eukaryotic Transcription • 3 different RNA polymerases – RNA polymerase I transcribes rRNA – RNA polymerase II transcribes mRNA and some snRNA – RNA polymerase III transcribes tRNA and some other small RNAs

• Each RNA polymerase recognizes its own promoter 11

• Initiation of transcription – Requires a series of transcription factors • Necessary to get the RNA polymerase II enzyme to a promoter and to initiate gene expression • Interact with RNA polymerase to form initiation complex at promoter

• Termination – Termination sites not as well defined

12

Other transcription factors

RNA polymerase II

Eukaryotic DNA

Transcription factor

Initiation complex

TATA box 1. A transcription factor recognizes and binds to the TATA box sequence, which is part of the core promoter.

2. Other transcription factors are recruited, and the initiation complex begins to build.

3. Ultimately, RNA polymerase II associates with the transcription factors and the DNA, forming the initiation complex, and transcription begins.

13

mRNA modifications • In eukaryotes, the primary transcript must be modified to become mature mRNA – Addition of a 5′ cap • Protects from degradation; involved in translation initiation

– Addition of a 3′ poly-A tail • Created by poly-A polymerase; protection from degradation

– Removal of non-coding sequences (introns) • Pre-mRNA splicing done by spliceosome 14

5′cap HO

OH

P

P CH2

P

+

3′

N+ CH3

Methyl group

P 5′

P

P

mRNA

CH3

15

Eukaryotic pre-mRNA splicing • Introns – non-coding sequences • Exons – sequences that will be translated • Small ribonucleoprotein particles (snRNPs) recognize the intron–exon boundaries • snRNPs cluster with other proteins to form spliceosome – Responsible for removing introns 16

E1

I1

E2

I2

E3

I3

DNA template

E4

I4

Exons Introns

Transcription 5' cap

3' poly-A tail

Primary RNA transcript Introns are removed 3' poly-A tail

5' cap

a.

Mature mRNA

Intron 1 mRNA 3

2

4 DNA

7 5

6 Exon

b.

c.

17

Alternative splicing • Single primary transcript can be spliced into different mRNAs by the inclusion of different sets of exons • 15% of known human genetic disorders are due to altered splicing • 35 to 59% of human genes exhibit some form of alternative splicing • Explains how 25,000 genes of the human genome can encode the more than 80,000 different mRNAs 18

tRNA and Ribosomes • tRNA molecules carry amino acids to the ribosome for incorporation into a polypeptide – Aminoacyl-tRNA synthetases add amino acids to the acceptor stem of tRNA – Anticodon loop contains 3 nucleotides complementary to mRNA codons

19

2D “Cloverleaf” ” Model

3D Ribbon-like Model Acceptor end

Acceptor end 3‫׳‬ 5‫׳‬

Anticodon loop

3D Space-filled Model Acceptor end

Anticodon loop

Anticodon loop

Icon Acceptor end

Anticodon end

20

tRNA charging reaction • Each aminoacyl-tRNA synthetase recognizes only 1 amino acid but several tRNAs • Charged tRNA – has an amino acid added using the energy from ATP – Can undergo peptide bond formation without additional energy

• Ribosomes do not verify amino acid attached to tRNA 21

Amino group NH3+ ATP

Pi Pi tRNA site

Carboxyl group Trp

C

Charged tRNA travels to ribosome

O

O– Amino acid site

NH3+

Trp

Accepting site

C

O

O

OH tRNA

Charged tRNA dissociates

Aminoacyl-tRNA Anticodon synthetase specific to tryptophan 1. In the first step of the reaction, the amino acid is activated. The amino acid reacts with ATP to produce an intermediate with the carboxyl end of the amino acid attached to AMP. The two terminal phosphates (pyrophosphates) are cleaved from ATP in this reaction.

2. The amino acid-AMP complex remains bound to the enzyme. The tRNA next binds to the enzyme.

3. The second step of there action transfers the amino acid from AMP to the tRNA, Producing a charged tRNA and AMP. The charged tRNA consists of a specific amino acid attached to the 3′ accept or stem of it sRNA.

22

• The ribosome has multiple tRNA binding sites – P site – binds the tRNA attached to the growing peptide chain – A site – binds the tRNA carrying the next amino acid – E site – binds the tRNA that carried the last amino acid 23

Large subunit

3′

Small subunit

Large subunit

90° °

Small subunit

Large subunit 0° °

Small subunit

mRNA 5′

24

• The ribosome has two primary functions – Decode the mRNA – Form peptide bonds

• Peptidyl transferase – Enzymatic component of the ribosome – Forms peptide bonds between amino acids

25

Translation • In prokaryotes, initiation complex includes – Initiator tRNA charged with N-formylmethionine – Small ribosomal subunit – mRNA strand

• Ribosome binding sequence (RBS) of mRNA positions small subunit correctly • Large subunit now added • Initiator tRNA bound to P site with A site empty 26

fMet

3′

AUG U A C

Initiation factor

mRNA

3′

Initiation factor

5′ Small subunit

GTP

5′

GDP +

Pi Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

tRNA in P site

U A C A U G

Large subunit

E site

A site

3′ 3′

GTP

GDP +

5′

5´´

Pi Initiation complex

Complete ribosome

27

• Initiations in eukaryotes similar except – Initiating amino acid is methionine – More complicated initiation complex – Lack of an RBS – small subunit binds to 5′ cap of mRNA

28

• Elongation adds amino acids – 2nd charged tRNA can bind to empty A site – Requires elongation factor called EF-Tu to bind to tRNA and GTP – Peptide bond can then form – Addition of successive amino acids occurs as a cycle 29

NH3+ Amino group Amino acid 1

NH3+

Polypeptide chain

NH3+

N

3′ O

O

NH3+ Amino acid 1

Peptide bond

Amino acid 2

Amino acid 2

Amino acid 2

Amino acid 1

Amino end (N terminus)

Peptide bond formation

“Empty” tRNA

OH

Amino acid 3 Amino acid 4

O

Amino acid 5 Amino acid 6 Amino acid 7

5′ COO– A site P site

Carboxyl end (C terminus)

30

3′

GDP

+

Pi

Elongati factor E

A

5′

factor

GTP

P

3′

3′

E

P

A

E

A

P

5′ 5′

Sectioned ribosome GTP

GTP Elongation factor

Next round

“Ejected” tRNA

3′

3′

E

P

Elongation factor GDP + Pi

Growing polypeptide

A E

P

A

5′ 5′

31

• There are fewer tRNAs than codons • Wobble pairing allows less stringent pairing between the 3′ base of the codon and the 5′ base of the anticodon • This allows fewer tRNAs to accommodate all codons

32

• Termination – Elongation continues until the ribosome encounters a stop codon – Stop codons are recognized by release factors which release the polypeptide from the ribosome

33

Polypeptide chain releases Dissociation

3′

Release factor

5′ 3′

5′ Sectioned ribosome A P E

34

Protein targeting • In eukaryotes, translation may occur in the cytoplasm or the rough endoplasmic reticulum (RER) • Signal sequences at the beginning of the polypeptide sequence bind to the signal recognition particle (SRP) • The signal sequence and SRP are recognized by RER receptor proteins • Docking holds ribosome to RER • Beginning of the protein-trafficking pathway 35

Rough endoplasmic reticulum (RER)

Cytoplasm

Lumen of the RER

Protein channel

SRP binds to signal peptide, arresting elongation Signal recognition particle (SRP)

Docking

NH2 Polypeptide elongation continues

Signal Exit tunnel

Ribosome synthesizing peptide

36

37

Mutation: Altered Genes • Point mutations alter a single base • Base substitution – substitute one base for another – Silent mutation – same amino acid inserted – Missense mutation – changes amino acid inserted • Transitions • Transversions

– Nonsense mutations – changed to stop codon 38

C G

A A T T

Coding

5′–ATGCCTTATCGCTGA–3′

Template

3′–TACGGAATAGCGACT–5′

mRNA

5′–AUGCCUUAUCGCUGA–3′

Protein

Met Pro Thr Arg Stop

a. Silent Mutation C G Coding

5′–ATGCCCTATCGCTGA–3′

Template mRNA

3′–TACGGGATAGCGACT–5′

Protein b.

5′–AUGCCCUAUCGCUGA–3′ Met Pro Thr

Arg Stop 39

Missense Mutation A T Coding

5′–ATGCCCTATCACTGA–3′

Template

3′–TACGGGATAGTGACT–5′ 5′–AUGCCCUAUCACUGA–3′

mRNA Protein

Met Pro Thr His Stop

c. Nonsense Mutation A T Coding

5′–ATGCCCTAACGCTGA–3′

Template

3′–TACGGGATTGCGACT–5′

mRNA

5′–AUGCCCUAACGCUGA–3′

Protein d.

Met Pro Stop 40

Normal Deoxygenated Tetramer

Normal HBB Sequence Polar Leu

C

T

Thr

G

A

C

Pro

T

C

C

Glu

T

G

A

Glu

G

A

A

Lys

G

A

A

Ser

G

T

C

Abnormal Deoxygenated Tetramer

Amino acids

T Nucleotides

1

2

1 2

1

2

1 2

Hemoglobin tetramer

"Sticky" nonpolar sites

Abormal HBB Sequence Nonpolar (hydrophobic) Leu

C

T

Thr

G

A

C

val

Pro

T

C

C

T

G

T

Lys

Glu

G

G

A

G

A

A

Ser

G

T

C

Amino acids

T Nucleotides

Tetramers form long chains when deoxygenated. This distorts the normal red blood cell shape into a sickle shape.

41

• Frameshift mutations – Addition or deletion of a single base – Much more profound consequences – Alter reading frame downstream – Triplet repeat expansion mutation • Huntington disease • Repeat unit is expanded in the disease allele relative to the normal

42

Chromosomal mutations • Change the structure of a chromosome – Deletions – part of chromosome is lost – Duplication – part of chromosome is copied – Inversion – part of chromosome in reverse order – Translocation – part of chromosome is moved to a new location

43

Deletion Deleted AB C D E F G H I J

AE F G H I J

a. Duplication Duplicated A B C D E F G H I J

A B C D B C D E F G H I J

b.

44

Inversion Inverted A B C D E F G H I J

A D C B E F G H I J

c. Reciprocal Translocation A B C D E F G H I J

K L M D E F G H I J

K L M N O P Q R

A B C N O P Q R

d.

45

• Mutations are the starting point for evolution • Too much change, however, is harmful to the individual with a greatly altered genome • Balance must exist between amount of new variation and health of species

46

Control of Gene Expression • Controlling gene expression is often accomplished by controlling transcription initiation • Regulatory proteins bind to DNA – May block or stimulate transcription

• Prokaryotic organisms regulate gene expression in response to their environment • Eukaryotic cells regulate gene expression to maintain homeostasis in the organism 47

Regulatory Proteins • Gene expression is often controlled by regulatory proteins binding to specific DNA sequences – Regulatory proteins gain access to the bases of DNA at the major groove – Regulatory proteins possess DNA-binding motifs

48

Vantage point

= Hydrogen bond donors = Hydrogen bond acceptors = Hydrophobic methyl group = Hydrogen atoms unable to form hydrogen bonds

DNA molecule 1

H N

H N

Phosphate

G

O

H

N

H

N

N

H

C

H N

N N

H

O

H

DNA molecule 2

H H

Phosphate

N

N

A

N

H

N

H

O

N

H

T N

N Sugar

CH3

H

O

49

Prokaryotic regulation • Control of transcription initiation – Positive control – increases frequency of initiation of transcription • Activators enhance binding of RNA polymerase to promoter • Effector molecules can enhance or decrease

– Negative control – decreases frequency • Repressors bind to operators in DNA • Allosterically regulated • Respond to effector molecules – enhance or abolish binding to DNA

50

• Prokaryotic cells often respond to their environment by changes in gene expression • Genes involved in the same metabolic pathway are organized in operons • Induction – enzymes for a certain pathway are produced in response to a substrate • Repression – capable of making an enzyme but does not 51

lac operon • Contains genes for the use of lactose as an energy source -galactosidase (lacZ), permease (lacY), and transacetylase (lacA)

• Gene for the lac repressor (lacI) is linked to the rest of the lac operon

52

CAP-binding site Gene for repressor protein Promoter for I gene

PI

I

CAP

Operator Promoter for lac operon

Plac

Gene for permease Genefor ß-galactosidase

Gene for transacetylase

O Z Y

A

Regulatory region Coding region lac Control system

53

• The lac operon is negatively regulated by a repressor protein – lac repressor binds to the operator to block transcription – In the presence of lactose, an inducer molecule (allolactose) binds to the repressor protein – Repressor can no longer bind to operator – Transcription proceeds

54

trp operon • Genes for the biosynthesis of tryptophan • The operon is not expressed when the cell contains sufficient amounts of tryptophan • The operon is expressed when levels of tryptophan are low • trp repressor is a helix-turn-helix protein that binds to the operator site located adjacent to the trp promoter 55

• The trp operon is negatively regulated by the trp repressor protein – trp repressor binds to the operator to block transcription – Binding of repressor to the operator requires a corepressor which is tryptophan – Low levels of tryptophan prevent the repressor from binding to the operator

56

Tryptophan Absent, Operon Derepressed E Inactive trp repressor (No tryptophan present)

D Translation

C B...