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The genetic code

Nucleic acids

Nucleic acids

Amino acids

Correspondence = the genetic code

Codon = triplet of three bases which encodes an amino acid

64 possible codons = 43 each of 4 nucleotides can occupy each of 3 positions in the codon

Deciphering the code

61 codons encode amino acids, 3 codons do not specify amino acids

Specialized codons:- for start of translation - AUG- for STOP - UAA, UAG, UGA

61 codons encode 20 amino acids - most amino acids are specified by more than one codon- degeneracy of the genetic code

Transfer RNA (tRNA) is the adapter tRNA has two crucial properties:

- it caarries a single amino acid to which it is covalently linked- it contains the anticodon (complementary to the codon representing its amino acid)

Codon-anticodon interactions

often one tRNA can recognize more than one codontRNALys can recognize AAA or AAG

3’ 5’

CGG

GCUmRNA 5’ 3’

anticodon

wobble hypothesis:the pairing between codon and anticodon at the first two codonpositions always follows the usual rules, but exceptional “wobbles”occur at the third position

Base in first positionof anticodon

Base recognized in thirdposition of codon

UCAG

A/G G UC/U

Structure of tRNA

Enzyme

ATP siteAmino acid site

tRNA site

Aminoacyl-tRNA synthetases

all synthetases function by two-step mechanism:1) activation of amino acid with ATP2) transfer of activated amino acid to tRNA

tRNA synthetases are responsible for the fidelity of translation

O-

-O-P=O

O-O-P=O

O-O-P=O

O

Adenosine

R

H-C-NH2

C

O O-

R

H-C-NH2

C

O O-

O-

P=O

O

AdenosineO-H

R

H-C-NH2

C

O O

Ribosome - site of protein synthesis

• ribosome provides the environment for controlling the interaction between mRNA and aminoacyl-tRNA

Ribosomes Subunits rRNA Proteins

50S

30S70S

Bacteria 23S, 5S

16S 21

31

60S

40S

80S

Mammals

28S, 5.8S, 5S

18S

49

33

5’ 3’Ribosome movement

The ribosome has two sites for binding charged tRNA

P-site = peptide sitegrowing peptide held by tRNA

A-site = acceptor siteentered by aminoacyl-tRNA

mRNA is associated with small (30S) subunit

tRNA spans both subunits amino acid end in the large subunit anticodon in the small subunit 5’ 3’

5’ 3’

5’ 3’

5’ 3’

Overview of protein synthesis

Before protein synthesistRNA with growing peptide in the P site;aminoacyl-tRNA in the A-site

Peptide bond formationInvolves transfer of polypeptide frompeptidyl-tRNA in P-site to aminoacyl-tRNAin A-site

TranslocationMoves ribosome one codon; placespeptidyl-tRNA in P-site; deacylated tRNAleaves the ribosome; A site is empty

Translation Initiation

Initiation - reactions before the first peptide bond formation - in prokaryotes always begins with free 30S subunits - formation of an initiation complex

50S

30S

Initiation Elongation Termination

Translation Initiation

Initiation occurs at a special sequence on mRNA- ribosome binding site (RBS) or Shine-Dalgarno sequence- complementary to the 3’end of 16S rRNA

5’ NNNNNAGGAGGU-N5-10-AUG---- 3’

3’ A U UCCUCCA

5’

mRNA

3’ end of 16S rRNA

Shine-Dalgarno sequence

Initiationcodon

Initiation codon- signal for initiation of translation- usually the triplet AUG (in bacteria also GUG or UUG)- AUG represents methionine

Translation Initiation

A special initiator tRNA starts the polypeptide chain- N-formyl-methionine tRNA - unique to bacteria- used only for initiation

NH2 O

H-C-----C-O CH2

CH2

S CH3

NH2 O

H-C-----C-O CH2

CH2

S CH3

OH-C-O

methionine N - formyl - methionine

Initiation requires initiation factors- found only on 30S subunit; released when 50S joins- three factors needed for mRNA and tRNA to enter the complex

5’ 3’AUGRBS

mRNA

IF3

5’ 3’AUGRBS

P

IF35’ 3’AUG

P

fMet

IF2

IF3

IF3

IF2

5’ 3’AUG

P

fMet

A

30S subunit

tRNAfMet

50S subunit

IF2

A-site is ready to accept any aminoacyl-tRNAexcept initiator tRNA

Chain elongation

5’ 3’

5’ 3’

EF

Elongation requires elongation factors and GTP

5’ 3’

EF

Peptidyl transferase reaction

5’3’

R

CH

HN C

O

OPeptide chain

5’3’

R

CH

N C

O

O R

CH

HN C

HO

OPeptide chain

R

CH

2HN C

O

O

Peptidyl-tRNA now in the A-site

Translocation moves the ribosome

ribosome advances three nucleotides along the mRNA

5’ 3’

result - expel the uncharged tRNA from the P-site - new peptidyl-tRNA enters P-site

- A-site is free for the next aminoacyl-tRNA or termination

Translation termination

5’ 3’

Dissociation

5’ 3’STOP

Release factor

3 triplets not represented by a tRNA: UAG, UAA, UGA

STOP codons are recognized by release factors (RF1, RF2)

Antibacterial antibiotics

Antibiotic Site of action

Streptomycin inhibits translation initiation; binds 30S subunitChloramphenicol inhibits elongation during translation; binds 50STetracycline inhibits translation; prevents aminoacyl tRNA bindingKanamycin inhibits translation; binds 30S and prevents

translocation

Rifamycin inhibits RNA synthesis; binds to ’ subunit of RNApolymerase

Novobiocin inhibits DNA gyrase

Ampicillin/Penicillin inhibits cell wall synthesis

Bacteria Eukaryotic cells

- mRNA transcribed and translated in the same compartment

- transcription and translation occursimultaneously

- mRNA is usually unstable - translatedfor short period of time (minutes)

- mRNA is usually polycistronic

- synthesis and maturation of mRNAoccur in the nucleus

- translation occurs in the cytoplasm

- mRNA is stable - translated forseveral hours

- mRNA is mostly monocistronic

RBS RBS

AUG AUG

STOP STOP

Intercistronicspacer

cap AAAAAAA

cap AAAAAAA

and promoters

Factor Gene Use -35 Sequence Separation -10 Sequence

70 rpoD general TGACA 16-18 bp TATAAT

32 rpoH heat shock CNCTTGAA 13-15 bp CCCCATNT

54 rpoN nitrogen CTGGNA 6 bp TTGCA

E. coli sigma factors recognize promoters with differentconsensus sequences