ch13 lecture genetics

7/28/2019 Ch13 Lecture GENETICS

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Chapter 13

TRANSLATION OF mRNA

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13.1 THE GENETIC BASIS FOR PROTEIN

SYNTHESIS

The translation of the mRNA codons into aminoacid sequences leads to the synthesis ofproteins

Proteins are the active participants in cell structureand function

Genes that encode polypeptides are termedstructural genes These are transcribed into messenger RNA (mRNA)

The main function of the genetic material is toencode the production of cellular proteins In the correct cell, at the proper time, and in suitable

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In the 1940s, Beadle and Tatum were interested inthe relationship between genes, enzymes and traits

They specifically asked this question

Is it One geneone enzyme or one genemany enzymes?

Their genetic model was Neurospora crassa (a

common bread mold)

Their studies involved the analysis of simple nutritional

requirements

Beadle and Tatums Experiments

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They analyzed more than 2,000 strains that had

been irradiated to produce mutations

They analyzed enzyme pathways for synthesis ofvitamins and amino acids

Figure 13.2 shows an example of their findings onthe synthesis of the amino acidArginine


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Figure 13.2

Every mutant strain was blocked at a particular step in the

synthesis pathway, showing that each gene encodedone enzyme

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In the normal strains, arginine was synthesized by

cellular enzymes

In the mutant strains, a genetic defect in one gene

prevented the synthesis of one protein required in onestep of the pathway to produce that amino acid

Beadle and Tatums conclusion: A single gene

controlled the synthesis of a single enzyme This was referred to as the one geneone enzyme

hypothesis


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Translation involves an interpretation of onelanguage into another

In genetics, the nucleotide language of mRNA is

translated into the amino acid language of proteins

Translation relies on the genetic code

Refer to Table 13.1

The genetic information is coded within mRNA in

groups ofthree nucleotides known as codons

The Genetic Code

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Figure 13.3

Figure 13.3 provides an overview of gene expression

Note that the start codon sets the

reading frame for all remaining codons

5

Template strand

Coding strand

Transcription

3

Translation

DNA

mRNA

tRNAPolypeptide

5 untranslated

region

3untranslated

region

Start

codon

Codons Anticodons

3

3

5

5

A C T G C C C A T G G G G C TC G A CA G GC G G G A A T A A C C G T C G A G G

G G C A G C T C C

C C G U C G A G G

T T GC A C

T G A C G G G T A C C C C G AG C T GT C CG C C C T T A T TA A CG T G

5 3A C U G C C C A U G G G G C UC G A CA G GC G G G A A U A AU U GC A C

Met Gly LeuSer Asp Gly GluHis Leu

Stop

codon

UAC CCC GAGUCG CUG CCC CUUGUG A AC

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Multiple codons may encode

the same amino acid.

These are known assynonymous codons

Three codons do not

encode an amino acid.

These are read as STOP

signals for translation

A code of 3 nucleotides could code for a

maximum of43 or64 amino acids; there are

20 standard amino acids 10


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Specialcodons:

AUG (which specifies methionine) = start codon

This defines the reading frame for all following codons AUG specifies additional methionines within the coding sequence

UAA, UAG and UGA = termination, orstop, codons

The code is degenerate More than one codon can specify the same amino acid

For example: GGU, GGC, GGA and GGG all code forglycine

In most instances, the third base is the variable base

It is sometime referred to as the wobble base

The code is nearly universal

Only a few rare exceptions have been noted

Refer to Table 13.3 11


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Polypeptide synthesis has a directionality that

parallels the 5 to 3 orientation of mRNA

During protein synthesis, a peptide bond is formed

between the carboxyl group of the last amino acid in

the polypeptide chain and the amino group in theamino acid being added

The first amino acid has an exposed amino group

Said to be N-terminal or amino terminal end The last amino acid has an exposed carboxyl group

Said to be C-terminal or carboxy terminal end

Refer to Figure 13.6

A Polypeptide Chain Has Directionality

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Peptide bond formation

H 3 N+ O

H HO

H3C

Amino

terminalend

Carboxyl

terminalend

Methionine Serine

Peptide bonds

Sequence in mRNA

Valine

CH2

CH3

CH3

CH2

CH2

OH

CH

S

C C CN

H

O

C CN C

H O H

Cysteine

CH2

SH

CN

H

O

C

Tyrosine

CH2

OH

H

CN C

H O

5 3A U G A G C GU U U A C U G C

H

Figure 13.6 Directionality in a polypeptide and mRNA14


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Figure 13.7

There are 20 amino acids that may be found in polypeptides Each contains a different side chain, orR group Each R group has its own particular chemical properties

Nonpolar amino acids arehydrophobic They are often buried within the

interior of a folded protein

H

H

Glycine (Gly) G

(a) Nonpolar, aliphatic amino acids

H3N C COO

CH3 CH3

CH

H

Alanine (Ala) A

H3N COO

CH3 CH3

CH

CH2

H

Valine (Val) V

H3N C COO

+

CH2CH2

CH2

H

Proline (Pro) P

H2N C COO

+

CH2

CH3

CH3 CH

H

Leucine (Leu) L Methionine (Met) M

H3N C COO

+

Cysteine (Cys) C

+

CH2

SH

H

H3N C COO

CH2

CH2

CH3

S

H

H3N C COO

+

H

Isoleucine (Ile) I

H3N C COO

+

(b) Aromatic amino acids

Phenylalanine (Phe) F Tyrosine (Tyr) Y

H

H3N C COO

+CH2

H

H3N C COO

+CH2

OH

Tryptophan (Trp) W

H

H3N C COO

+CH2

N

H

+

CH3

C

+

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There are four levels of structure in proteins

1. Primary

2. Secondary

3. Tertiary 4. Quaternary

A proteins primary structure is its amino acid

sequence Refer to Figure 13.8

Levels of Structure in Proteins

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Lys

NH3+

110

20

30

40

50

60

70

80

90

100

110

120

129

ValPhe Gly

Arg Cys GluLeu

Ala

Ala

Ala

Met

Lys

Arg

His

GlyLeuAsp

AsnTyrArgGlyTyr

Ser

Thr

AspTyr

GlyLeu

Asn

SerGluPheLysAlaAlaCysValTrpAsn

LeuGly

Phe

Asn

ThrGinAla

ThrAsnArgAsn

Thr

Asp

Gly

Ser

lle

Glnlle

AsnSer

Arg Trp Trp

Cys

Asn

AspGly

ArgThrProGlySer

ArgAsnLeuCys

Asn

lle

Pro

CysSer Ala Leu

LeuSer

SerAsp

lleThr

Arg AsnArg

Cys

Lys

Gly

Thr

Asp

AlaTrp ValAla

Asn

Met

GlyAsp

GlyAsp Ser Val lle Lys Lys Ala

CysAsn

Val

Ser

Ala

ValGlnAlaTrplleArgGlyCys

Arg

Leu

Trp

COO

Figure 13.8

The amino acid

sequence of the

enzyme

lysozyme

129 amino acids

long

Within the cell, theprotein will not be foundin this linear state

Rather, it will adopt a

compact 3-Dstructure

Indeed, this foldingcan begin during

translation

The progression fromthe primary structure tothe 3-D structure is

dictated by the aminoacid sequence withinthe polypeptide

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Figure 13.9

helix

sheet

Primary

structure

Secondary

structure

Quaternary

structure

Tertiary

structure

Proteinsubunit

AlaC

O

C

C

C

C

O

O

Val

Phe

Glu

Tyr

Leu

Iso

Ala

H

N

NH3+

NH3+

COO

COO

NH3+

COO

H

N

CC

CC O

O

HH

NN

H

N

CC

C

CC

CO

OC

O

H

H

N

NN

Depending onthe amino acidsequence,some regionsmay fold intoan helix or sheet.

Two or morepolypeptidesmay associatewith each other.

Regions ofsecondarystructure andirregularly shapedregions fold into athree-dimensionalconformation.

(a)

(b)

(c)

(d)

H

Levels of Structures in Proteins

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The primary structure of a protein folds to formregular, repeating shapes known as secondary

structures

There are two types of secondary structures a helix

b sheet

Refer to Figure 13.9


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The short regions of secondary structure in a protein

fold into a three-dimensional tertiary structure This is the final conformation of proteins that are

composed of a single polypeptide

Structure determined by hydrophobic and ionic interactions as well as

hydrogen bonds and Van der Waals interactions

Proteins made up oftwo or more polypeptides have

a quaternary structure

This is formed when the various polypeptides associate

with one another to make a functional protein


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tRNA has two functions1. Recognizing a 3-base codon in mRNA

2. Carrying an amino acid that is specific for that codon

During mRNA-tRNA recognition, the anticodon in tRNA binds to a

complementary codon in mRNA

13.2 STRUCTURE AND FUNCTION OF tRNA

tRNAs are named

according to the

amino acid they bear

The anticodon isanti-parallel to

the codon

Phenylalanine

tRNAPhe tRNAPro

Phenylalanineanticodon

Phenylalaninecodon

Prolinecodon

A G

Proline

Prolineanticodon

U C

3 mRNA5

G CA G

U C C G

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RNA Sh C S l F


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tRNAs Share Common Structural Features

The secondary structure of

tRNAs exhibits a cloverleafpattern; It contains 3 stem-loop structures

A few variable sites

An acceptor stem with a 3 singlestrand region

In addition to the normalnucleotides, tRNAs commonly

contain 80 modified nucleotides

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The enzymes that attach amino acids to tRNAs are

known as aminoacyl-tRNA synthetases

There are 20 types

One for each amino acid

Aminoacyl-tRNA synthetases catalyze a two-step

reaction involving three different molecules

Amino acid, tRNA and ATP

Refer to the figure next slide

Charging of tRNAs

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The amino acid is attached to the 3 end of the tRNA by an ester bond

Charging of tRNAs

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13.3 RIBOSOME STRUCTURE AND

ASSEMBLY

Translation occurs on the surface of the ribosome

Bacterial cells have one type of ribosome

Found in the cytoplasm

Eukaryotic cells have two types of ribosomes One type is found in the cytoplasm

The other is found in organelles

Mitochondria ; Chloroplasts

A ribosome is composed of structures called the large

and small subunits

Each subunit is formed from the assembly of

Proteins

rRNA

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13 3 RIBOSOME STRUCTURE AND


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13.3 RIBOSOME STRUCTURE AND

ASSEMBLY

A ribosome is composed of structures called the large and small subunits27


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During bacterial translation, the mRNA lies on the

surface of the 30S subunit As a polypeptide is being synthesized, it exits through a

channel within the 50S subunit

Ribosomes contain three discrete sites Peptidyl site (P site)

Aminoacyl site (A site)

Exit site (E site)

Functional Sites of Ribosomes

Model for ribosome structure

Polypeptide

30S

50S

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tRNA

mRNA

E P A

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13 4 STAGES OF TRANSLATION


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mRNA

UACAnticodon

InitiatortRNA tRNA

with firstamino acid

AUGStart codon

AUGStart codon

UAGStop codon

UAGStop codon

Completedpolypeptide

3. Termination

2. Elongation(This stepoccurs manytimes.)

Recycling of translationalcomponents

Releasefactor

Small

LargeRibosomalsubunits

EEA

E AP

aa1aa2aa3aa4

aa5

aa1aa1

33 55

35

35

P P A

Figure 13.16

Initiator tRNA

1. Initiation

13.4 STAGES OF TRANSLATION

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The mRNA, initiator tRNA, and ribosomal subunits

associate to form an initiation complex

This process requires three Initiation Factors

The initiator tRNA recognizes the start codon in

mRNA

In bacteria, this tRNA is designated tRNAfmet

It carries a methionine that has been covalently modified toN-formyl-methionine

The start codon is AUG

The Translation Initiation Stage

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Shine-Dalgarno

sequence

mRNA

5 3A U C U A G U A A G U U C A GG G U CG A GU C A C G C A GU G GG U A

3

Start

codon

A U U C C C AC 16S rRNAU

The binding of mRNA to the 30S subunit is facilitated by a

ribosomal-binding site orShine-Dalgarno sequence

This is complementary to a sequence in the 16S rRNA

Figure 13.18

Hydrogen bonding

Component of the

30S subunit

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Figure 13.17

IF2, which uses GTP, promotesthe binding of the initiator tRNAto the start codon in the P site.

Portion of16S rRNA

The mRNA binds to the 30S subunit.The Shine-Dalgarno sequence iscomplementary to a portion of the16S rRNA.

IF1 and IF3 bind to the 30S subunit.

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30S subunit

Shine-Dalgarnosequence

(9 nucleotideslong)

Startcodon

IF3 IF1

IF1IF3

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tRNAfMet


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Figure 13.17

70S

initiation

complex

This marks

the end of the

initiation

stage

IF1 and IF3 are released.

IF2 hydrolyzes its GTP and is released.

The 50S subunit associates.

tRNAfMet

IF2GTP

E AP

35

35

70Sinitiationcomplex

IF1IF3

Initiator tRNA

tRNAfMet

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In eukaryotes, the assembly of the initiation complex

is similar to that in bacteria

However, additional factors are required

Note that eukaryotic Initiation Factors are denoted eIF

Refer to Table 13.7

The initiator tRNA is designated tRNAmet It carries a methionine rather than a formylmethionine

The Translation Initiation Stage

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The start codon for eukaryotic translation is AUG

Ribosome scans from the 5 end of mRNA until it finds

the AUG start codon (not all AUGs can act as a start)

The consensus sequence for optimal start codonrecognition is shown here

Start codon

G C C (A/G) C C A U G G-6 -5 -4 -3 -2 -1 +1 +2 +3 +4

Most important positions for codon selection

These rules are called Kozaks rules

After Marilyn Kozak who first proposed them

With that in mind, the start codon for eukaryotic

translation is usually the first AUG after the 5 Cap!

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Translational initiation in eukaryotes can be

summarized as such:

An initiation factor protein complex (eIF4) binds to the 5

cap in mRNA

These are joined by a complex consisting of the 40S

subunit, tRNAmet, and other initiation factors The entire assembly moves along the mRNA scanning

for the right start codon

Once it finds this AUG, the 40S subunit binds to it

The 60S subunit joins This forms the 80S initiation complex

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During this stage, amino acids are added to the

polypeptide chain, one at a time

This process, though complex, can occur at a

remarkable rate In bacteria 15-20 amino acids per second

In eukaryotes 2-6 amino acids per second

The Translation Elongation Stage

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Figure 13.19

The 23S rRNA (a

component of the large

subunit) is the actual

peptidyl transferase

Thus, the ribosome

is a ribozyme!

3

P site

Codon 3Codon 4

mRNA

E siteA site

aa1aa2

aa3 Ribosome

aa1aa2aa3

EAP

aa4

A charged tRNA bindsto the A site. EF-Tufacilitates tRNA bindingand hydrolyzes GTP.

Peptidyltransferase, which

is a component of the 50Ssubunit, catalyzes peptidebond formation between thepolypeptide and the aminoacid in the A site. Thepolypeptide is transferred

to the A site.

5

5

3

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Figure 13.19

tRNAs at the P and

A sites move into

the E and P sites,

respectively

Codon 4Codon 5

Codon 335

aa1aa2aa3aa4

aa1aa2

aa3

E A

A

Codon 4

Codon 5Codon 33

5

aa1aa2aa3

aa4

E

A

P

P

aa4

This process is repeated, again and

again, until a stop codon is reached.

An unchargedtRNA is releasedfrom the E site.

The ribosome translocates1 codon to the right. This

translocation is promotedby EF-G, which hydrolyzesGTP.

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E P

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16S rRNA (a part of the 30S ribosomal subunit) plays

a key role in codon-anticodon recognition

If incorrect tRNA bound at the A site

It will prevent elongation until the mispaired tRNA is released

This phenomenon is termed the decoding function

of the ribosome

It is important in maintaining the high fidelity of mRNAtranslation

Error rate: 1 mistake per 10,000 amino acids added

The Translation Elongation Stage

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The final stage occurs when a stop codon is

reached in the mRNA

In most species there are three stop ornonsense codons

UAG UAA

UGA

These codons are not recognized by tRNAs, but byproteins called release factors

The 3-D structure of release factors mimics that of

tRNAs

The Translation Termination Stage

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Bacteria have three release factors

RF1, which recognizes UAA and UAG

RF2, which recognizes UAA and UGA RF3, which does not recognize any of the three codons

It binds GTP and helps facilitate the termination process

Eukaryotes only have one release factor eRF, which recognizes all three stop codons

The Translation Termination Stage

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Figure 13.20

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Stop codonin A site

tRNA in Psite carriescompletedpolypeptide

E A

35

E A

mRNA A release factor (RF) binds to the A site.

The polypeptide is cleaved from the tRNAin the P site. The tRNA is then released.

The ribosomal subunits, mRNA, andrelease factor dissociate.

Releasefactor

3

3

5

5

50S subunit 30S subunit

mRNA

P

P

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Bacteria lack a nucleus

Therefore, both transcription and translation occur in the

cytoplasm

As soon an mRNA strand is long enough, a ribosome will

attach to its 5 end

So translation begins before transcription ends

This phenomenon is termed coupling

A polyribosome orpolysome is an mRNA transcript that has

many bound ribosomes in the act of translation

Bacterial Translation Can Begin

Before Transcription Is Completed

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Coupling between transcription and translation in bacteria

ch13 lecture genetics

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