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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
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
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