Genetics, Lecture 6, Translation 1 (Slides)

8/8/2019 Genetics, Lecture 6, Translation 1 (Slides)

1/16

Protein Synthesis and the Genetic Code

a). Overview of translation

i). Requirements for protein synthesis

ii). messenger RNA

iii). Ribosomes and polysomes

iv). Polarity of protein synthesis

b). Transfer RNA

i). tRNA as an adaptorii). Amino acid activation

iii). Aminoacyl tRNA synthetases

iv). Charged tRNA

c). The genetic code

i). Codon-anticodon interactionsii). Initiation codon in prokaryotes vs. eukaryotes

iii). Reading frame

d). Mutations affecting translation

i). Frameshift mutations

ii). Missense and nonsense mutations


2/16

Learning Objectives

Understand the structure of the ribosome and polysome

Understand the role of tRNA as an "adaptor" Understand the mechanism for charging tRNAs

Understand the general characteristics of the genetic code Know the start and stop codons Understand the mechanism for how some tRNAs can recognize more

than one codon Understand how the AUG initiation codon is recognized in prokaryotes

and eukaryotes

Understand how the AUG codon establishes the reading frame Know the various kinds of mutations that affect the translation of

proteins


3/16

Overview of translation

last step in the flow of genetic information definition of translation requirements for protein synthesis

mRNA ribosomes initiation factors

elongation and termination factors GTP aminoacyl tRNAs

amino acids aminoacyl tRNA synthetases ATP


4/16

Messenger RNA (mRNA)

m7Gppp

Cap

55 untranslated region

AUG

initiationcodon

translated (coding) region

(AAAA)npoly(A)

tail

3 untranslated region

UGAtermination

codon

3AAUAAA


5/16

Ribosomes prokaryotic ribosome

eukaryotic ribosome

70S ribosome

80S ribosome

50S subunit23S rRNA

5S rRNA

35 proteins

60S subunit28S rRNA

5S rRNA

5.8S rRNA

49 proteins

30S subunit16S rRNA

21 proteins

40S subunit18S rRNA

33 proteins


6/16

Polysomes direction of translation is 5 to 3 along the mRNA direction of protein synthesis is N terminus to C terminus

UGA5

large ribosomal subunit

small ribosomal subunit

AUG

polysome

nascent

polypeptide

NN

subunits dissociate


7/16

Transfer RNA tRNA is the adaptor molecule in protein synthesis acceptor stem

CCA-3 terminus to which amino acid is coupled

carries amino acid on terminal adenosineanticodon stem and anticodon loop


8/16

Amino acid activation and aminoacyl tRNA synthetases

aminoacyl tRNA synthetases are the enzymes that charge the tRNAs 20 amino acids one aminoacyl tRNA synthetase for each amino acid can be several different isoacceptor tRNAs for each amino acid all isoacceptor tRNAs for an amino acid use the same synthetase

each aminoacyl tRNA synthetase binds amino acid ATP

isoacceptor tRNAs


9/16

H2N-C-C-OH

H

R-

-

O=

ATP

H2N-C-C-O-P-O-ribose-adenine

H

R-

-

O=

amino acid

adenylated (activated)

amino acid

PPi

uncharged tRNA

H2N-C-C-OH

R-

-

O=

aminoacyl

(charged)

tRNA

AMP

3

Amino acid activation

and

tRNA charging


10/16

The genetic code

consists of 64 triplet codons (A, G, C, U) 43 = 64

all codons are used in protein synthesis 20 amino acids 3 termination (stop) codons: UAA, UAG, UGA

AUG (methionine) is the start codon (also used internally)

multiple codons for a single amino acid = degeneracy

5 amino acids are specified by the first two nucleotides only

3 additional amino acids (Arg, Leu, and Ser) are specified by

six different codons


11/16

The Genetic Code

UUU

UUCUUA

UUG

CUU

CUC

CUACUG

AUU

AUC

AUAAUG

GUU

GUC

GUA

GUG

UCU

UCCUCA

UCG

CCU

CCC

CCACCG

ACU

ACC

ACAACG

GCU

GCC

GCA

GCG

UAU

UACUAA

UAG

CAU

CAC

CAACAG

AAU

AAC

AAAAAG

GAU

GAC

GAA

GAG

UGU

UGCUGA

UGG

CGU

CGC

CGACGG

AGU

AGC

AGAAGG

GGU

GGC

GGA

GGG

Phe

Leu

Leu

Val

Ile

Met

Ser

Pro

Thr

Ala

Tyr

Stop

His

Gln

Asn

Lys

Asp

Glu

Cys

Arg

Ser

Arg

Gly

Stop

Trp


12/16

Codon-anticodon interactions codon-anticodon base-pairing is antiparallel the third position in the codon is frequently degenerate one tRNA can interact with more than one codon (therefore 50 tRNAs) wobble rules

C with G or I (inosine) A with U or I G with C or U U with A, G, or I I with C, U, or A

5 3

A U G

U A C

3 5 tRNAmet

mRNA

5 3

C U A

G

G A U

3 5 tRNAleu

mRNA

wobble base

one tRNAleu can read two

of the leucine codons


13/16

Inosine = Cytidine Inosine = Adenosine

Inosine = Uridine Guanosine = Uridine

Wobble Interactions


14/16

Initiation in prokaryotes and eukaryotes initiation can occur at internal AUG codons in prokaryotic mRNA initiation in eukaryotes occurs only at the first AUG codon

lac operon in E. coli is transcribed as a polycistronic mRNA

with multiple AUG codons

eukaryotic mRNA

lac I P O lac Z lac Y lac A

AUG AUG AUG

AUGSD AUGSDAUG

initiation codon with

Shine-Dalgarno site

initiation codon with

Shine-Dalgarno site

internal Met codon

does not have

Shine-Dalgarno site

5

5 cap AUG

initiation can only occur at

first AUG codon downstream of the 5 cap

AUG

internal (downstream) Met codon

cannot serve as an initiation site

AUG


15/16

Reading frame reading frame is determined by the AUG initiation codon every subsequent triplet is read as a codon until reaching a stop codon

...AGAGCGGA.AUG.GCA.GAG.UGG.CUA.AGC.AUG.UCG.UGA.UCGAAUAAA...

MET.ALA.GLU.TRP.LEU.SER.MET.SER

a frameshift mutation

...AGAGCGGA.AUG.GCA.GA .UGG.CUA.AGC.AUG.UCG.UGA.UCGAAUAAA...

the new reading frame results in the wrong amino acid sequence andthe formation of a truncated protein

...AGAGCGGA.AUG.GCA.GAU.GGC.UAA.GCAUGUCGUGAUCGAAUAAA...

MET.ALA.ASP.GLY


16/16

Mutations affecting translation hemoglobin Wayne (3 terminal frameshift mutation)

Normal -globin .ACG.UCU.AAA.UAC.CGU.UAA.GCU GGA GCC UCG GUA.THR.SER.LYS.TYR.ARG

Wayne -globin .ACG.UCA.AAU.ACC.GUU.AAG.CUG.GAG.CCU.CGG.UAG.THR.SER.ASN.THR.VAL.LYS.LEU.GLU.PRO.ARG

mutated region

missense mutations (e.g., AGC Ser to AGA Arg) nonsense mutations (e.g., UGG Trp to UGA Stop)

read through, reverse terminator, or sense mutations(e.g., UAA Stop to CAA Gln) as in hemoglobin Constant Spring

silent mutations (e.g., CUA Leu to CUG Leu) do not affect translation

Documents

Genetics, Lecture 6, Translation 1 (Slides)