The genetic code Codon  = triplet of three bases which encodes an amino acid 64 possible codons = 4 3   each of 4 nucleoti...
Deciphering the code <ul><li>61 codons encode amino acids, 3 codons do not specify amino acids </li></ul><ul><li>Specializ...
Codon-anticodon interactions <ul><li>often one tRNA can recognize more than one codon </li></ul><ul><ul><li>tRNA Lys  can ...
Structure of tRNA
Aminoacyl-tRNA synthetases <ul><li>all synthetases function by two-step mechanism: </li></ul><ul><ul><li>1) activation of ...
Ribosome - site of protein synthesis <ul><li>ribosome provides the environment for controlling the interaction between </l...
The ribosome has two sites for binding charged tRNA <ul><li>mRNA is associated with small (30S) subunit </li></ul><ul><li>...
Overview of protein synthesis Before protein synthesis tRNA with growing peptide in the P site; aminoacyl-tRNA in the A-si...
Translation Initiation <ul><li>Initiation - reactions before the first peptide bond formation </li></ul><ul><ul><ul><li>- ...
Translation Initiation <ul><li>Initiation occurs at a special sequence on mRNA </li></ul><ul><ul><li>- ribosome binding si...
Translation Initiation <ul><li>A special initiator tRNA starts the polypeptide chain </li></ul><ul><ul><li>- N-formyl-meth...
IF3 IF3 IF2 30S subunit tRNAf Met 50S subunit IF2 5’ 3’ AUG RBS mRNA IF3 5’ 3’ AUG RBS P IF3 5’ 3’ AUG P fMet IF2 5’ 3’ AU...
Chain elongation EF <ul><li>Elongation requires elongation factors and GTP </li></ul>5’ 3’ 5’ 3’ 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...
Translocation moves the ribosome <ul><li>ribosome advances three nucleotides along the mRNA </li></ul><ul><li>result - exp...
Translation termination Dissociation Release factor <ul><li>3 triplets not represented by a tRNA:  UAG, UAA, UGA </li></ul...
Antibacterial antibiotics Antibiotic Site of action Streptomycin inhibits translation initiation; binds 30S subunit Chlora...
Bacteria Eukaryotic cells -  mRNA transcribed and translated  in the same compartment -  transcription and translation occ...
   and promoters
<ul><li>E. coli  sigma factors recognize promoters with different </li></ul><ul><li>consensus sequences </li></ul>Factor  ...
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  1. 1. 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 Nucleic acids Nucleic acids Amino acids Correspondence = the genetic code
  2. 2. Deciphering the code <ul><li>61 codons encode amino acids, 3 codons do not specify amino acids </li></ul><ul><li>Specialized codons: </li></ul><ul><ul><li>- for start of translation - AUG </li></ul></ul><ul><ul><li>- for STOP - UAA, UAG, UGA </li></ul></ul><ul><li>61 codons encode 20 amino acids </li></ul><ul><li>- most amino acids are specified </li></ul><ul><li> by more than one codon </li></ul><ul><li>- degeneracy of the genetic code </li></ul>Transfer RNA (tRNA) is the adapter <ul><li>tRNA has two crucial properties: </li></ul><ul><ul><li>- it caarries a single amino acid to which it is covalently linked </li></ul></ul><ul><ul><li>- it contains the anticodon (complementary to the codon </li></ul></ul><ul><ul><li>representing its amino acid) </li></ul></ul>
  3. 3. Codon-anticodon interactions <ul><li>often one tRNA can recognize more than one codon </li></ul><ul><ul><li>tRNA Lys can recognize AAA or AAG </li></ul></ul><ul><li>wobble hypothesis: </li></ul><ul><ul><li>the pairing between codon and anticodon at the first two codon </li></ul></ul><ul><ul><li>positions always follows the usual rules, but exceptional “wobbles” </li></ul></ul><ul><ul><li>occur at the third position </li></ul></ul>3’ 5’ CGG GCU mRNA 5’ 3’ anticodon Base in first position of anticodon Base recognized in third position of codon U C A G A/G G U C/U
  4. 4. Structure of tRNA
  5. 5. Aminoacyl-tRNA synthetases <ul><li>all synthetases function by two-step mechanism: </li></ul><ul><ul><li>1) activation of amino acid with ATP </li></ul></ul><ul><ul><li>2) transfer of activated amino acid to tRNA </li></ul></ul><ul><li>tRNA synthetases are responsible for the fidelity of translation </li></ul>Enzyme ATP site Amino acid site tRNA site 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 O
  6. 6. Ribosome - site of protein synthesis <ul><li>ribosome provides the environment for controlling the interaction between </li></ul><ul><li>mRNA and aminoacyl-tRNA </li></ul>Ribosomes Subunits rRNA Proteins 50S 30S 70S Bacteria 23S, 5S 16S 21 31 60S 40S 80S Mammals 28S, 5.8S, 5S 18S 49 33
  7. 7. The ribosome has two sites for binding charged tRNA <ul><li>mRNA is associated with small (30S) subunit </li></ul><ul><li>tRNA spans both subunits </li></ul><ul><ul><li>amino acid end in the large subunit </li></ul></ul><ul><ul><li>anticodon in the small subunit </li></ul></ul>5’ 3’ Ribosome movement P-site = peptide site growing peptide held by tRNA A-site = acceptor site entered by aminoacyl-tRNA 5’ 3’
  8. 8. 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 5’ 3’ 5’ 3’ 5’ 3’
  9. 9. Translation Initiation <ul><li>Initiation - reactions before the first peptide bond formation </li></ul><ul><ul><ul><li>- in prokaryotes always begins with free 30S subunits </li></ul></ul></ul><ul><ul><ul><li>- formation of an initiation complex </li></ul></ul></ul>50S 30S Initiation Elongation Termination
  10. 10. Translation Initiation <ul><li>Initiation occurs at a special sequence on mRNA </li></ul><ul><ul><li>- ribosome binding site (RBS) or Shine-Dalgarno sequence </li></ul></ul><ul><ul><li>- complementary to the 3’end of 16S rRNA </li></ul></ul><ul><li>Initiation codon </li></ul><ul><ul><li>- signal for initiation of translation </li></ul></ul><ul><ul><li>- usually the triplet AUG (in bacteria also GUG or UUG) </li></ul></ul><ul><ul><li>- AUG represents methionine </li></ul></ul>5’ NNNNNAGGAGGU-N 5-10 -AUG---- 3’ 3’ A U UCCUCCA 5’ mRNA 3’ end of 16S rRNA Shine- Dalgarno sequence Initiation codon
  11. 11. Translation Initiation <ul><li>A special initiator tRNA starts the polypeptide chain </li></ul><ul><ul><li>- N-formyl-methionine tRNA - unique to bacteria </li></ul></ul><ul><ul><li>- used only for initiation </li></ul></ul><ul><li>Initiation requires initiation factors </li></ul><ul><ul><li>- found only on 30S subunit; released when 50S joins </li></ul></ul><ul><ul><li>- three factors needed for mRNA and tRNA to enter the complex </li></ul></ul>NH 2 O H-C-----C-O CH 2 CH 2 S CH3 NH 2 O H-C-----C-O CH 2 CH 2 S CH3 O H-C-O methionine N - formyl - methionine
  12. 12. IF3 IF3 IF2 30S subunit tRNAf Met 50S subunit IF2 5’ 3’ AUG RBS mRNA IF3 5’ 3’ AUG RBS P IF3 5’ 3’ AUG P fMet IF2 5’ 3’ AUG P fMet A A-site is ready to accept any aminoacyl-tRNA except initiator tRNA
  13. 13. Chain elongation EF <ul><li>Elongation requires elongation factors and GTP </li></ul>5’ 3’ 5’ 3’ 5’ 3’ EF
  14. 14. 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
  15. 15. Translocation moves the ribosome <ul><li>ribosome advances three nucleotides along the mRNA </li></ul><ul><li>result - expel the uncharged tRNA from the P-site </li></ul><ul><li>- new peptidyl-tRNA enters P-site </li></ul><ul><ul><li>- A-site is free for the next aminoacyl-tRNA or termination </li></ul></ul>5’ 3’
  16. 16. Translation termination Dissociation Release factor <ul><li>3 triplets not represented by a tRNA: UAG, UAA, UGA </li></ul><ul><li>STOP codons are recognized by release factors (RF1, RF2) </li></ul>5’ 3’ 5’ 3’ STOP
  17. 17. Antibacterial antibiotics Antibiotic Site 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  ’ subunit of RNA polymerase Novobiocin inhibits DNA gyrase Ampicillin/Penicillin inhibits cell wall synthesis
  18. 18. Bacteria Eukaryotic 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 RBS AUG AUG STOP STOP Intercistronic spacer cap AAAAAAA cap AAAAAAA
  19. 19.  and promoters
  20. 20. <ul><li>E. coli sigma factors recognize promoters with different </li></ul><ul><li>consensus sequences </li></ul>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

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