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The genetic code

Nucleic acids. Nucleic acids. Correspondence = the genetic code. Amino acids. 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.

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The genetic code

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  1. Nucleic acids Nucleic acids Correspondence = the genetic code Amino acids 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

  2. 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)

  3. 3’ 5’ anticodon CGG GCU mRNA 5’ 3’ Base in first position of anticodon Base recognized in third position of codon U C A G A/G G U C/U Codon-anticodon interactions • often one tRNA can recognize more than one codon tRNALys can recognize AAA or AAG • 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

  4. Structure of tRNA

  5. Enzyme Amino acid site ATP site R H-C-NH2 C O O- O- -O-P=O O -O-P=O O -O-P=O O Adenosine R H-C-NH2 C O O O- P=O O Adenosine O-H tRNA site R H-C-NH2 C O O- Aminoacyl-tRNA synthetases • all synthetases function by two-step mechanism: 1) activation of amino acid with ATP 2) transfer of activated amino acid to tRNA • tRNA synthetases are responsible for the fidelity of translation

  6. Ribosome - site of protein synthesis • ribosome provides the environment for controlling the interaction between • mRNA and aminoacyl-tRNA Ribosomes Subunits rRNA Proteins 50S 23S, 5S 31 Bacteria 70S 16S 21 30S Mammals 60S 28S, 5.8S, 5S 49 80S 40S 33 18S

  7. P-site = peptide site growing peptide held by tRNA A-site = acceptor site entered by aminoacyl-tRNA 3’ 3’ 5’ 5’ Ribosome movement The ribosome has two sites for binding charged tRNA • mRNA is associated with small (30S) subunit • tRNA spans both subunits amino acid end in the large subunit anticodon in the small subunit

  8. 3’ 3’ 3’ 5’ 5’ 5’ 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

  9. 30S 50S Initiation Elongation Termination Translation Initiation • Initiation - reactions before the first peptide bond formation - in prokaryotes always begins with free 30S subunits - formation of an initiation complex

  10. 3’ end of 16S rRNA 3’ A U UCCUCCA 5’ 5’ NNNNNAGGAGGU-N5-10-AUG---- 3’ mRNA Shine- Dalgarno sequence Initiation codon 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 • Initiation codon • - signal for initiation of translation • - usually the triplet AUG (in bacteria also GUG or UUG) • - AUG represents methionine

  11. O H-C-O NH2 O H-C-----C-O CH2 CH2 S CH3 NH2 O H-C-----C-O CH2 CH2 S CH3 methionine N - formyl - methionine Translation Initiation • A special initiator tRNA starts the polypeptide chain • - N-formyl-methionine tRNA - unique to bacteria • - used only for initiation • Initiation requires initiation factors • - found only on 30S subunit; released when 50S joins • - three factors needed for mRNA and tRNA to enter the complex

  12. RBS RBS mRNA 5’ 5’ 3’ 3’ AUG AUG A-site is ready to accept any aminoacyl-tRNA except initiator tRNA IF3 P fMet IF2 3’ 5’ AUG IF3 P fMet 3’ 5’ AUG A P 30S subunit IF3 tRNAfMet IF2 50S subunit IF3 IF2

  13. 3’ 3’ 3’ 5’ 5’ 5’ EF Chain elongation • Elongation requires elongation factors and GTP EF

  14. 3’ 3’ 5’ 5’ R CH HN C O R CH N C O R CH 2HN C O O O O Peptide chain R CH HN C HO O Peptide chain Peptidyl-tRNA now in the A-site Peptidyl transferase reaction

  15. 3’ 5’ Translocation moves the ribosome • ribosome advances three nucleotides along the mRNA • 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

  16. 3’ 3’ 5’ 5’ STOP Translation termination • 3 triplets not represented by a tRNA: UAG, UAA, UGA • STOP codons are recognized by release factors (RF1, RF2) Release factor Dissociation

  17. Antibacterial antibiotics AntibioticSite of action Streptomycin inhibits translation initiation; binds 30S subunit Chloramphenicol inhibits elongation during translation; binds 50S Tetracycline inhibits translation; prevents aminoacyl tRNA binding Kanamycin inhibits translation; binds 30S and prevents translocation Rifamycin inhibits RNA synthesis; binds to b’ subunit of RNA polymerase Novobiocin inhibits DNA gyrase Ampicillin/Penicillin inhibits cell wall synthesis

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

  19. sand promoters

  20. Factor Gene Use -35 Sequence Separation -10 Sequence s70rpoD general TGACA 16-18 bp TATAAT s32rpoH heat shock CNCTTGAA 13-15 bp CCCCATNT s54rpoN nitrogen CTGGNA 6 bp TTGCA • E. coli sigma factors recognize promoters with different consensus sequences

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