The genetic code Nucleic acids Amino acids Correspondence = the genetic code Codon = triplet of three bases which encodes an amino acid 64 possible codons = 4 3 each of 4 nucleotides can occupy each of 3 positions in the codon

Deciphering the code 4 61 codons encode amino acids, 3 codons do not specify amino acids 4 Specialized codons: - for start of translation - AUG - for STOP - UAA, UAG, UGA 4 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 4 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 4 often one tRNA can recognize more than one codon tRNA Lys can recognize AAA or AAG 3’5’ CGG GCU mRNA 5’ 3’ anticodon 4 wobble hypothesis: the pairing between codon and anticodon at the first two codon positions always follows the usual rules, but exceptional “wobbles” occur at the third position Base in first position of anticodon Base recognized in third position of codon UCAGUCAG A/G G U C/U

Structure of tRNA

Enzyme ATP site Amino acid site tRNA site Aminoacyl-tRNA synthetases 4 all synthetases function by two-step mechanism: 1) activation of amino acid with ATP 2) transfer of activated amino acid to tRNA 4 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-NH 2 C O O- R H-C-NH 2 C O O- O - P=O O Adenosine O-H R H-C-NH 2 C O

Ribosome - site of protein synthesis ribosome provides the environment for controlling the interaction between mRNA and aminoacyl-tRNA RibosomesSubunitsrRNAProteins 50S 30S 70S Bacteria 23S, 5S 16S S 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 site growing peptide held by tRNA A-site = acceptor site entered by aminoacyl-tRNA 4 mRNA is associated with small (30S) subunit 4 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 synthesis tRNA with growing peptide in the P site; aminoacyl-tRNA in the A-site Peptide bond formation Involves transfer of polypeptide from peptidyl-tRNA in P-site to aminoacyl-tRNA in A-site Translocation Moves ribosome one codon; places peptidyl-tRNA in P-site; deacylated tRNA leaves the ribosome; A site is empty

Translation Initiation 4 Initiation - reactions before the first peptide bond formation - in prokaryotes always begins with free 30S subunits - formation of an initiation complex 50S 30S InitiationElongationTermination

Translation Initiation 4 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-N AUG---- 3’ 3’ A U UCCUCCA 5’ mRNA 3’ end of 16S rRNA Shine- Dalgarno sequence Initiation codon 4 Initiation codon - signal for initiation of translation - usually the triplet AUG (in bacteria also GUG or UUG) - AUG represents methionine

Translation Initiation 4 A special initiator tRNA starts the polypeptide chain - N-formyl-methionine tRNA - unique to bacteria - used only for initiation NH 2 O H-C-----C-O CH 2 S CH3 NH 2 O H-C-----C-O CH 2 S CH3 O H-C-O methionineN - formyl - methionine 4 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’ AUG RBS mRNA IF3 5’3’ AUG RBS P IF3 5’ 3’ AUG P fMet IF2 IF3 IF2 5’ 3’ AUG P fMet A 30S subunit tRNAf Met 50S subunit IF2 A-site is ready to accept any aminoacyl-tRNA except initiator tRNA

Chain elongation 5’ 3’ 5’ 3’ EF 4 Elongation requires elongation factors and GTP 5’ 3’ EF

Peptidyl transferase reaction 5’ 3’ R CH HN C O O Peptide chain 5’ 3’ R CH N C O O R CH HN C HO O Peptide chain R CH 2 HN C O O Peptidyl-tRNA now in the A-site

Translocation moves the ribosome 4 ribosome advances three nucleotides along the mRNA 5’ 3’ 4 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 4 3 triplets not represented by a tRNA: UAG, UAA, UGA 4 STOP codons are recognized by release factors (RF1, RF2)

Antibacterial antibiotics AntibioticSite of action Streptomycininhibits translation initiation; binds 30S subunit Chloramphenicolinhibits elongation during translation; binds 50S Tetracyclineinhibits translation; prevents aminoacyl tRNA binding Kanamycininhibits translation; binds 30S and prevents translocation Rifamycininhibits RNA synthesis; binds to  ’ subunit of RNA polymerase Novobiocininhibits DNA gyrase Ampicillin/Penicillininhibits cell wall synthesis

BacteriaEukaryotic cells - mRNA transcribed and translated in the same compartment - transcription and translation occur simultaneously - mRNA is usually unstable - translated for short period of time (minutes) - mRNA is usually polycistronic - synthesis and maturation of mRNA occur in the nucleus - translation occurs in the cytoplasm - mRNA is stable - translated for several hours - mRNA is mostly monocistronic RBS AUG STOP Intercistronic spacer cap AAAAAAA cap AAAAAAA

 and promoters

Factor Gene Use -35 Sequence Separation -10 Sequence  70 rpoD general TGACA bp TATAAT  32 rpoH heat shock CNCTTGAA bp CCCCATNT  54 rpoN nitrogen CTGGNA 6 bp TTGCA 4 E. coli sigma factors recognize promoters with different consensus sequences