n chapter 12~ from gene to protein. protein synthesis: overview n one gene-one enzyme hypothesis...

Chapter 12~ From Gene to Protein

Protein Synthesis: overview One gene-one enzyme

hypothesis (Beadle and Tatum)

One gene-one polypeptide (protein) hypothesis

Transcription: synthesis of RNA under the direction of DNA (mRNA)

Translation: actual synthesis of a polypeptide under the direction of mRNA

Figure 17.3 The triplet code

The Central Dogma of Molecular Biology

4

codon 1 codon 2

nontemplate strand

template strand

codon 3

polypeptide N N NC C C C C C

R1 R2 R3

Serine Aspartate Proline

O O O

C

G

G

CA

G

CC

G

C

GTT

AG

C G

C

G CA CC CAG C

transcriptionin nucleus

translationat ribosome

mRN A

35

35

53

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

DNA

The Genetic Code of Life

RNA is a polymer of RNA nucleotides– RNA nucleotides contain the sugar ribose instead of

deoxyribose– RNA nucleotides are of four types: uracil (U),

adenine (A), cytosine (C), and guanine(G)– Uracil (U) replaces thymine (T) of DNA

Types of RNA• Messenger (mRNA) - Takes a message from DNA in the

nucleus to ribosomes in the cytoplasm• Ribosomal (rRNA) - Makes up ribosomes, which read the

message in mRNA• Transfer (tRNA) - Transfers the appropriate amino acid to

the ribosomes for protein synthesis5

Structure of RNA

6


G

U

A

C

S

S

S

S

P

P

P

P

base isuracil insteadof thymine

one nucleotideribose3′ end

5′ end

RNA Structure Compared to DNA structure

7

The Genetic Code of Life The unit of the genetic code consists of codons,

each of which is a unique arrangement of symbols Each of the 20 amino acids found in proteins is

uniquely specified by one or more codons – The symbols used by the genetic code are the mRNA bases

• Function as “letters” of the genetic alphabet• Genetic alphabet has only four “letters” (U, A, C, G)

– Codons in the genetic code are all three bases (symbols) long

• Function as “words” of genetic information• Permutations:

– There are 64 possible arrangements of four symbols taken three at a time

– Often referred to as triplets• Genetic language only has 64 “words”

8

Messenger RNA Codons

9

U C A G

U

C

A

G

U

C

A

G

U

C

A

G

U

C

A

G

U

C

A

G

Second Base ThirdBase

FirstBase

CGAarginine

CGGarginine

AGUserine

AGCserine

AGAarginine

AGGarginine

GGUglycine

GGCglycine

GGAglycine

GGGglycine

UGGtryptophan

CGUarginine

CGCarginine

UGAstop

AUG (start)methionine

CAChistidine

CAAglutamine

CAGglutamine

AAUasparagine

AACasparagine

AAAlysine

AAGlysine

GAUaspartate

GACaspartate

GAAglutamate

GAGglutamate

UUUphenylalanine

CUUleucine

CUCleucine

CUAleucine

CUGleucine

AUUisoleucine

AUCisoleucine

AUAisoleucine

GUUvaline

GUCvaline

GUAvaline

GUGvaline

UCAserine

CCUproline

CCCproline

CCAproline

CCGproline

ACUthreonine

ACCthreonine

ACAthreonine

ACGthreonine

GCUalanine

GCCalanine

GCAalanine

GCGalanine

CAUhistidine

UAAstop

UCUserine

UAUtyrosine

UGUcysteine

UUCphenylalanine

UCCserine

UACtyrosine

UGUcysteine

UUAleucine

UCGserine

UAGstop

UUGleucine


The Genetic Code of Life

Properties of the genetic code:– Universal

• With few exceptions, all organisms use the code the same way• Encode the same 20 amino acids with the same 64 triplets

– Degenerate (redundant)• There are 64 codons available for 20 amino acids• Most amino acids encoded by two or more codons

– Unambiguous (codons are exclusive)• None of the codons code for two or more amino acids• Each codon specifies only one of the 20 amino acids

– Contains start and stop signals• Punctuation codons• Like the capital letter we use to signify the beginning of a

sentence, and the period to signify the end

10

12.4 First Step: Transcription Transcription

– A segment of DNA serves as a template for the production of an RNA molecule

– The gene unzips and exposes unpaired bases– Serves as template for mRNA formation– Loose RNA nucleotides bind to exposed DNA

bases using the C=G and A=U rule– When entire gene is transcribed into mRNA, the

result is a pre-mRNA transcript of the gene– The base sequence in the pre-mRNA is

complementary to the base sequence in DNA

11

First Step: Transcription

A single chromosome consists of one very long molecule encoding hundreds or thousands of genes

The genetic information in a gene describes the amino acid sequence of a protein– The information is in the base sequence of one side (the “sense”

strand) of the DNA molecule– The gene is the functional equivalent of a “sentence”

The segment of DNA corresponding to a gene is unzipped to expose the bases of the sense strand– The genetic information in the gene is transcribed (rewritten) into an

mRNA molecule– The exposed bases in the DNA determine the sequence in which the

RNA bases will be connected together– RNA polymerase connects the loose RNA nucleotides together

The completed transcript contains the information from the gene, but in a mirror image, or complementary form

12

Transcription

13


T

TG

C

A

T

T

G

C

A

terminator

gene strand

promoter

templatestrand

direction ofpolymerasemovement

RNA polymerase

DNA template strand

mRNAtranscript

to RNA processing

3′

3′5′

5′

5′

3′

RNA Polymerase

14© Oscar L. Miller/Photo Researchers, Inc.


a. 200m

b.

spliceosome

DNA

RNApolymerase

RNAtranscripts

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Animation


Pre-mRNA is modified before leaving the eukaryotic nucleus. – Modifications to the ends of the primary

transcript:• Cap on the 5′ end

– The cap is a modified guanine (G) nucleotide– Helps a ribosome determine where to attach when

translation begins

• Poly-A tail of 150-200 adenines on the 3′ end– Facilitates the transport of mRNA out of the nucleus– Inhibits degradation of mRNA by hydrolytic enzymes.

16

First Step: Transcription Pre-mRNA, is composed of exons and introns.

– The exons will be expressed, – The introns, occur in between the exons.

• Allows a cell to pick and choose which exons will go into a particular mRNA

– RNA splicing:• Primary transcript consists of:

– Some segments that will not be expressed (introns)

– Segments that will be expressed (exons)

• Performed by spliceosome complexes in nucleoplasm– Introns are excised

– Remaining exons are spliced back together

Result is a mature mRNA transcript17


In prokaryotes, introns are removed by “self-splicing”—that is, the intron itself has the capability of enzymatically splicing itself out of a pre-mRNA

18

Messenger RNA Processing in Eukaryotes

19


DNA


20


exon

intron intron

exon exonDNA


21


exon

intron intron

exon exonDNA

transcription

exon

intron intron

exon exonpre-mRNA

5 3


22

exon

intron intron

exon exon

transcription

exon

intron intron

exon exonpre-mRNA

5 3

exon

intron intron

exon exon

cap3 5

DNA

poly-A tail



23


exon

intron intron

exon exon

transcription

exon

intron intron

exon exonpre-mRNA

5 3

exon

intron intron

exon exon

exon exon exon

cap poly-A tail

spliceosome

cap

3

3

5

5

DNA

poly-A tail


24

exon

intron intron

exon exonDNA

transcription

exon

intron intron

exon exonpre-mRNA

5 3

exon

intron intron

exon exon

exon

intron RNA

exon

pre-mRNAsplicing

exon

cap poly-A tail

spliceosome

mRNA

cap poly-A tail

cap poly-A tail

3

3

3

5

5

5



25

exon

intron intron

exon exonDNA

transcription

exon

intron intron

exon exonpre-mRNA

5 3

exon

intron intron

exon exon

exon

intron RNA

exon

pre-mRNAsplicing

exon

cap poly-A tail

spliceosome

mRNA

cytoplasm

cap poly-A tail

cap poly-A tail

3

3

3

5

5

5



26

exon

intron intron

exon exonDNA

transcription

exon

intron intron

exon exon

5 3

exon

intron intron

exon exon

exon

intron RNA

exon

pre-mRNAsplicing

exon

cap poly-A tail

spliceosome

nucleus

mRNA

cytoplasm

nuclear porein nuclear envelope

cap poly-A tail

cap poly-A tail

3

3

5

5

pre-mRNA

3 5



Animation

First Step: Transcription Functions of introns:

– As organismal complexity increases;• The number of protein-coding genes does not keep pace• But the proportion of the genome that is introns increases

– Possible functions of introns:• Exons might combine in various combinations

– Would allow different mRNAs to result from one segment of DNA• Introns might regulate gene expression• Introns may encourage crossing-over during meiosis

– Exciting new picture of the genome is emerging

28

12.5 Second Step: Translation

Translation– The sequence of codons in the mRNA at a ribosome directs the sequence of

amino acids into a polypeptide– A nucleic acid sequence is translated into a protein sequence

tRNA molecules have two binding sites:– One associates with the mRNA transcript– The other associates with a specific amino acid– Each of the 20 amino acids in proteins associates with one or more of 64

types of tRNA Translation

– An mRNA transcript associates with the rRNA of a ribosome in the cytoplasm or a ribosome associated with the rough endoplasmic reticulum

– The ribosome “reads” the information in the transcript– Ribosome directs various types of tRNA to bring in their specific amino acid

“fares”– The tRNA specified is determined by the code being translated in the mRNA

transcript

29

Second Step: Translation tRNA molecules come in 64 different kinds All are very similar except that

– One end bears a specific triplet (of the 64 possible) called the anticodon

– The other end binds with a specific amino acid type– tRNA synthetases attach the correct amino acid to

the correct tRNA molecule All tRNA molecules with a specific anticodon

will always bind with the same amino acid

30

Messenger RNA Codons

31

U C A G

U

C

A

G

U

C

A

G

U

C

A

G

U

C

A

G

U

C

A

G

Second Base ThirdBase

FirstBase

CGAarginine

CGGarginine

AGUserine

AGCserine

AGAarginine

AGGarginine

GGUglycine

GGCglycine

GGAglycine

GGGglycine

UGGtryptophan

CGUarginine

CGCarginine

UGAstop

AUG (start)methionine

CAChistidine

CAAglutamine

CAGglutamine

AAUasparagine

AACasparagine

AAAlysine

AAGlysine

GAUaspartate

GACaspartate

GAAglutamate

GAGglutamate

UUUphenylalanine

CUUleucine

CUCleucine

CUAleucine

CUGleucine

AUUisoleucine

AUCisoleucine

AUAisoleucine

GUUvaline

GUCvaline

GUAvaline

GUGvaline

UCAserine

CCUproline

CCCproline

CCAproline

CCGproline

ACUthreonine

ACCthreonine

ACAthreonine

ACGthreonine

GCUalanine

GCCalanine

GCAalanine

GCGalanine

CAUhistidine

UAAstop

UCUserine

UAUtyrosine

UGUcysteine

UUCphenylalanine

UCCserine

UACtyrosine

UGUcysteine

UUAleucine

UCGserine

UAGstop

UUGleucine


3’

5’

hydrogenbonding

aminoacid

leucine

G A A

Structure of a tRNA Molecule

32



33


3’

5’

hydrogenbonding

amino acid end

aminoacid

leucine

G A A


34


G A A

3’

5’

hydrogenbonding

amino acid end

anticodon end

aminoacid

leucine

anticodon


35


G

GA

A A

CCC CC CU U UU

3’

5’

hydrogenbonding

amino acid end

anticodon end

mRNA5’ 3’

aminoacid

leucine

anticodon


36


G

GA

A A

CCC CC CU U UU

3’

5’

hydrogenbonding

amino acid end

anticodon end

mRNA5’ 3’

aminoacid

leucine

anticodon

a. b.codon

Second Step: Translation

Ribosomes

– Ribosomal RNA (rRNA):

• Produced from a DNA template in the nucleolus of a nucleus

• Combined with proteins into large and small ribosomal subunits

– A completed ribosome has three binding sites to facilitate pairing between tRNA and mRNA

• The E (for exit) site

• The P (for peptide) site, and

• The A (for amino acid) site37

Ribosome Structure and Function

38

tRNA bindingsites

outgoingtRNA

3

mRNA

incomingtRNA

polypeptide

5

small subunit

mRNA

large subunit

a. Structure of a ribosome b. Binding sites of ribosome

c. Function of ribosomes d. Polyribosome


Courtesy Alexander Rich

EP

A

39


Animation


Initiation:– Components necessary for initiation are:

• Small ribosomal subunit• mRNA transcript• Initiator tRNA, and• Large ribosomal subunit• Initiation factors (special proteins that bring the

above together)– Initiator tRNA:

• Always has the UAC anticodon• Always carries the amino acid methionine• Capable of binding to the P site

40


Small ribosomal subunit attaches to mRNA transcript

– Beginning of transcript always has the START codon (AUG)

Initiator tRNA (UAC) attaches to P site

Large ribosomal subunit joins the small subunit

41

Initiation

42

U A CA U G

U A CA U G

A small ribosomal subunitbinds to mRNA; an initiatortRNA pairs with the mRNAstart codon AUG. The large ribosomal subunit

completes the ribosome.Initiator tRNA occupies theP site. The A site is readyfor the next tRNA.

Met

amino acid methionine

initiator tRNA

mRNA

3

5

small ribosomal subunit

large ribosomal subunit

P site A siteE site

35

Met

Initiation

start codon


Second Step: Elongation “Elongation” refers to the growth in length of

the polypeptide RNA molecules bring their amino acid fares

to the ribosome– Ribosome reads a codon in the mRNA

• Allows only one type of tRNA to bring its amino acid• Must have the anticodon complementary to the

mRNA codon being read• The incoming tRNA joins the ribosome at its A site

– Methionine of the initiator tRNA is connected to the amino acid of the 2nd tRNA by a peptide bond

43

Second Step: Elongation The second tRNA moves to P site (translocation) The spent initiator tRNA moves to the E site and exits The ribosome reads the next codon in the mRNA

– Allows only one type of tRNA to bring its amino acid• Must have the anticodon complementary to the

mRNA codon being read• Joins the ribosome at its A site

– The dipeptide on the 2nd amino acid is connected to the amino acid of the 3rd tRNA by a peptide bond

44

Elongation

45


UA

C AUG G A C

35

peptidebond

Met

Ala

Trp

Ser

Val

Elongation

46

UA

C AUG

C U G

G A C

A tRNA–amino acidapproaches theribosome and bindsat the A site.

35

anticodon

tRNApeptidebond

Met

asp

Ala

Trp

Ser

Val

1


Elongation

UA

C AUG

C U G

G A CUA

C AUG G A C

C U G


Two tRNAs can be at aribosome at one time;the anticodons arepaired to the codons.

anticodon

tRNApeptidebond

Metasp

Ala

Trp

Ser

Val

353

5

Met

Ala

Trp

Ser

Val Asp

1 2


47

Elongation

48

UA

C AUG

C U G

G A C

UA

C AUG G A C

C U G


Two tRNAs can be at aribosome at one time;

the anticodons arepaired to the codons.

anticodon

tRNApeptide bond

Metasp

Ala

Trp

Ser

Val

Met

Ala

Trp

Ser

Val Asp

1 2

UA

C AUG G A C

C U G

Peptide bond formationattaches the peptidechain to the newlyarrived amino acid.

353

5 35

Met

Val

Asp

Ala

Trp

Ser

3

peptide bond


Elongation

49

U

A

C A

UG

C U G

G A C

U

A

C A

UG G A C

C U G



anticodon

tRNApeptidebond

asp

35 5

Asp

1 2 4

U

A

C A

UG G A C

C U G

UC

A

G A C

C U G

AUG A C C


The ribosome moves forward; the“empty” tRNA exits from the E site;the next amino acid–tRNA complexis approaching the ribosome.

35

Met

Val

Asp

Ala

Trp

Ser

3 35

Met

Val

Asp

Ala

Trp

Ser

3

peptidebond

Met

Ala

Trp

Ser

Val

Met

Ala

Trp

Ser

Val


Elongation

50

UA

C AUG

C U G

G A C

U

A

C A

UG G A C

C U G



anticodon

tRNApeptidebond

asp

35 5

Asp

1 2 4

UA

C A

UG G A C

C U G

UC

A

G A C

C U G

AUG

U G G

A C C



35

Met

Val

Asp

Ala

Trp

Ser

3 35

Met

Val

Asp

Ala

Trp

Ser Thr

3

peptidebond


Met

Ala

Trp

Ser

Val

Met

Ala

Trp

Ser

Val

Elongation

51

Elongation


UA

C AUG

C U G

G A C

U

A

C A

UG G A C

C U G



anticodon

tRNApeptidebond

asp

35 5

Asp

1 2 4

UA

C A

UG G A C

C U G

UC

A

G A C

C U G

AUG

U G G

A C C



35

Met

Val

Asp

Ala

Trp

Ser

3 35

Met

Val

Asp

Ala

Trp

Ser Thr

3

peptidebond

Met

Ala

Trp

Ser

Val

Met

Ala

Trp

Ser

Val

Second Step: Translation Termination:

– Previous tRNA moves to the P site– Spent tRNA moves to the E site and exits– Ribosome reads the STOP codon at the end of the

mRNA• UAA, UAG, or UGA• Does not code for an amino acid

– A protein called a release factor binds to the stop codon and cleaves the polypeptide from the last tRNA

– The ribosome releases the mRNA and dissociates into subunits

– The same mRNA may be read by another ribosome

52

Termination

53


The ribosome comes to a stopcodon on the mRNA. A releasefactor binds to the site.

The release factor hydrolyzes the bondbetween the last tRNA at the P site andthe polypeptide, releasing them. Theribosomal subunits dissociate.

release factorAla

Asp

GluUA

UA U G A

AG A

U G A

UC

U

Termination

stop codon

Asp

Ala

Trp

Trp

Val

Val

Glu

3′

5′

5′ 3′

Summary of Protein Synthesis in Eukaryotes

54

5

5

3mRNA

pre-mRNA

DNA

introns

6. During elongation, polypeptide synthesis takes place one amino acid at a time.

7. Ribosome attaches to rough ER. Polypeptide enters lumen, where it folds and is modified.

3. mRNA moves into cytoplasm and becomes associated with ribosomes.

4. tRNAs with anticodons carry amino acids to mRNA.

TRANSLATIONTRANSCRIPTION

nuclear pore

mRNA

large and smallribosomal subunits

aminoacids

anticodon

tRNA

peptide

codon

ribosome

5

3

3

C CG G

GCG

CG

C

CCC

GUA

CU A

UA

UA

UUA

UA

AU

CG

A

1. DNA in nucleus serves as a template for mRNA.

2. mRNA is processed before leaving the nucleus.

5. During initiation, anticodon-codon complementary base pairing begins as the ribosomal subunits come together at a start codon.

8. During termination, a ribosome reaches a stop codon; mRNA and ribosomal subunits disband.



Animation

12.6 Structure of the Eukaryotic Chromosome Each chromosome contains a single linear DNA

molecule, but is composed of more than 50% protein.

Some of these proteins are concerned with DNA and RNA synthesis

Histones play primarily a structural role– The most abundant proteins in chromosomes– Five primary types of histone molecules– Responsible for packaging the DNA

• The DNA double helix is wound at intervals around a core of eight histone molecules (called a nucleosome)

• Nucleosomes are joined by “linker” DNA.

56

Structure of Eukaryotic Chromosomes

57a: © Ada L. Olins and Donald E. Olins/Biological Photo Service; b: Courtesy Dr. Jerome Rattner, Cell Biology and Anatomy, University of Calgary; c: Courtesy of Ulrich Laemmli and J.R. Paulson, Dept. of Molecular Biology, University of Geneva, Switzerland; d: © Peter Engelhardt/Department of Pathology and Virology, Haartman Institute/Centre of Excellence in Computational Complex Systems Research,

Biomedical Engineering and Computational Science, Faculty of Information and Natural Sciences, Helsinki University of Technology, Helsinki, Finland


1. Wrapping of DNA around histone proteins.

2. Formation of a three-dimensional zigzag structure via histone H1 and other DNA-binding proteins.

3. Loose coiling into radial loops .

4. Tight compaction of radial loops to form heterochromatin.

5. Metaphase chromosome forms with the help of a protein scaffold.

DNAdouble helix

nucleosome

euchromatin

c. Radial loop domains

d. Heterochromatin

e. Metaphase chromosome

1,400 nm

700 nm

300 nm

30 nm

histone H1

histones

11 nm

2nm

a. Nucleosomes (“beads on a string”)

b. 30-nm fiber

Mutations: genetic material changes in a cell Point mutations: Changes in 1 or a few base pairs in

a single gene Base-pair substitutions:

– silent mutations no effect on protein – missense ∆ to a different amino acid (different

protein) – nonsense ∆ to a stop codon and a nonfunctional

protein Base-pair insertions or deletions: additions or losses

of nucleotide pairs in a gene; alters the ‘reading frame’ of triplets~frameshift mutation

Mutagens: physical and chemical agents that change DNA

Figure 17.23 The molecular basis of sickle-cell disease: a point mutation

Figure 17.24 Categories and consequences of point mutations: Base-pair insertion or deletion

Figure 17.24 Categories and consequences of point mutations: Base-pair substitution

n chapter 12~ from gene to protein. protein synthesis: overview n one gene-one enzyme hypothesis...

Documents

codons code

genetic code of lifeproperties

dnathe genetic code

genetic code of lifethe

synthesis of rna

amino acidsmost amino

messenger rna codons

structure of rna