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Proverbs 3:1-3 1 2 3 My Son, forget not my law; but let thine heart keep my commandments: For length of days, and long life, and peace, shall they add to thee. Let not mercy and truth forsake thee: bind them about thy neck; write them upon the table of thine heart: ©2001 Timothy G. Standish Messenger RNA Timothy G. Standish, Ph. D. ©2001 Timothy G. Standish Introduction The Central Dogma of Molecular Biology Cell Transcription Translation Reverse transcription DNA mRNA Ribosome Polypeptide (protein) Information Only Goes One Way The central dogma states that once “information” has passed into protein it cannot get out again. The transfer of information from nucleic acid to nucleic acid, or from nucleic acid to protein, may be possible, but transfer from protein to protein, or from protein to nucleic acid, is impossible. Information means here the precise determination of sequence, either of bases in the nucleic acid or of amino acid residues in the protein. Francis Crick, 1958 ©2001 Timothy G. Standish Transcription Coding (sense) strand 5’ 3’ 3’ 5’ Template (antisense) strand ©2001 Timothy G. Standish Transcription Coding (sense) strand 5’ 3’ 3’ 5’ 5’ RNA Pol. Template (antisense) strand RNA ©2001 Timothy G. Standish Transcription Coding (sense) strand 5’ 3’ 3’ 5’ Template (antisense) strand RNA Pol. 5’ ©2001 Timothy G. Standish RNA Polymerase RNA Polymerase is a spectacular enzyme, it performs the following functions: Recognition of the promoter region Melting of DNA (Helicase + Topisomerase) RNA Priming (Primase) RNA Polymerization Recognition of terminator sequence ©2001 Timothy G. Standish Products of Transcription Transcription produces three major RNA products: 1 Ribosomal RNA (rRNA) - Several rRNAs are vital constituents of ribosomes 2 Transfer RNA (tRNA) - The molecule that physically couples nucleic acid codons with specific amino acids 3 Messenger RNA (mRNA) - The nucleic acid messenger that carries encoded information from genes on DNA to the protein manufacturing ribosomes ©2001 Timothy G. Standish Stages of Transcription 1. 2. 3. Transcription can be logically divided into three distinct stages: Initiation Elongation Termination ©2001 Timothy G. Standish A “Simple” Gene Transcription Start Site 5’ 5’ Untranslated Region 3’ Untranslated Region Protein Coding Region 3’ RNA Transcript Promoter/ Control Region Terminator Sequence ©2001 Timothy G. Standish Transcription Initiation Proteins called transcription factors bind to the promoter region of a gene If the appropriate transcription factors are present, RNA polymerase binds to form an initiation complex RNA polymerase melts the DNA at the transcription start site Polymerization of RNA begins ©2001 Timothy G. Standish Initiation T. F. Promoter T. F. RNA Pol. RNA Pol. RNA 5’ ©2001 Timothy G. Standish Transcription Termination There are two types of termination: Rho dependent requires a protein called Rho, that binds to and slides along the RNA transcript. The terminator sequence slows down the elongation complex, Rho catches up and knocks it off the DNA Rho independent termination depends on both slowing down the elongation complex, and an AT-rich region that destabilizes the elongation complex ©2001 Timothy G. Standish Termination Rho Independent RNA Pol. RNA Pol. RNA 5’ RNA Pol. 5’ RNA 5’ Terminator ©2001 Timothy G. Standish Termination Rho Dependent Terminator RNA Pol. RNA 5’ 5’ r Help, Rho hit me! r RNA Pol. RNA Pol. RNA 5’ ©2001 Timothy G. Standish Differences Between Transcription In Prokaryotes and Eukaryotes ©2001 Timothy G. Standish Transcription And Translation In Prokaryotes 5’ 3’ 3’ 5’ RNA Pol. Ribosome mRNA Ribosome 5’ ©2001 Timothy G. Standish Eukaryotic Transcription Cytoplasm DNA Transcription RNA RNA Processing mRNA G G AAAAAA Nucleus AAAAAA Export ©2001 Timothy G. Standish A “Simple” Eukaryotic Gene Transcription Start Site 3’ Untranslated Region 5’ Untranslated Region Introns 5’ Exon 1 Int. 1 Promoter/ Control Region Exon 2 3’ Int. 2 Exon 3 Terminator Sequence Exons RNA Transcript 5’ Exon 1 Int. 1 Exon 2 Int. 2 Exon 3 3’ ©2001 Timothy G. Standish Processing Eukaryotic mRNA 5’ Untranslated Region 3’ Untranslated Region Protein Coding Region 5’5’ G 3’ Int. 11 Exon Int. 23 Exon 3 AAAAA 3’ Exon 1Exon Exon22 Exon 5’ Cap 3’ Poly A Tail RNA processing achieves three things: Removal of introns Addition of a 5’ cap Addition of a 3’ tail l This signals the mRNA is ready to move out of the nucleus and may control its lifespan in the cytoplasm ©2001 Timothy G. Standish The 5’ Cap 5’ End of hnRNA O H O P O H N O GTP H C C H C N C O C H C H C C O O H ©2001 Timothy G. Standish The 5’ Cap O H O C P O P O O P O H N N O C C O H N C OH H O H C H C O N C O C H C C C H O H O C C H H C H C C O H O H ©2001 Timothy G. Standish Transfer RNA (tRNA) Acts as the adapter molecule between the genetic code on mRNA and the protein “language” 75-85 bases long A specific amino acid is covalently linked at the 3’ end Elsewhere on the molecule is an anticodon complimentary to the specific amino acid codon on mRNA that codes for the amino acid carried by the tRNA Contain a number of modified bases ©2001 Timothy G. Standish Transfer RNA (tRNA) Acceptor Arm - A specific amino acid is attached to the 3’ end 16 Pu 17 9 A 17:1 13 12 Py 10 1 2 3 4 5 6 U* 7 Amino Acid A C C 73 72 71 70 69 68 67 Py 59A* 66 65 64 63 62 C D Arm - Contains dihydrouridine Py* TyC arm - y stands for pseudouridine Pu 49 50 51 52 G T C y Py G* 22 23 Pu 25 G 26 2020:120:2A 27 1 28 29 30 31 attachment site 47:16 47:15 43 44 42 45 41 46 47 40 47:1 39 38 Pu* U 34 35 36 Anticodon Extra Arm - May vary in size ©2001 Timothy G. Standish Aminoacyl-tRNA Synthetase Aminoacyl-tRNA Synthetase enzymes attach the correct amino acids to the correct tRNA This is an energy-consuming process Aminoacyl-tRNA Synthetases recognize tRNAs on the basis of their looped structure, not by direct recognition of the anticodon ©2001 Timothy G. Standish Gly P P Aminoacyl-tRNA Synthetase P ATP Gly P P P Aminoacyl-tRNA Synthetase Making AminoacyltRNA Pyrophosphate Gly P Aminoacyl-tRNA Synthetase CCA Gly P P Making AminoacyltRNA Aminoacyl-tRNA Synthetase P ATP Gly P P P Aminoacyl-tRNA Synthetase Pyrophosphate Gly Aminoacyl-tRNA Synthetase Gly Aminoacyl-tRNA Synthetase P AMP CCA AminoacyltRNA CCA Note that the amino acid is not paired with the tRNA on the basis of the anticodon. The correct tRNA for a given amino acid is recognized on the basis of other parts of the molecule. ©1998 Timothy G. Standish O H H N C Aminoacylation of tRNA O H C H O H H R C H O H C H H O H H C H C O N C 3’ 5’ H O O H O H C C C N C N P N C O H N H ©2001 Timothy G. Standish Class I Aminoacyl Aminoacylation O tRNA Synthetases attach amino acids to of tRNA C H H N Amino acid O C R H H C H O H C H H O H H C H N C 3’ 5’ O O N H C C C N C N P H O tRNA C O H the 2’ carbon while Class II attach to the 3’carbon C O H N H ©2001 Timothy G. Standish Classification of AminoacyltRNA Synthetases Aminoacyl-tRNA Class I - 2’ OH Class II - 3’ OH Synthetases Glu (a) Gly (a b2 Gln (a) Ala (a4 (ARS) may be Arg (a) Pro (a mono or Val (a) Ser (a multimeric. Ile (a) Thr (a Two types of Leu (a) Asp (a ?? polypeptide Met (a Asn (a chains are Tyr (a His (a recognized: (a Lys (a a and b. ©2001 Timothy G. Standish Requirements for Translation Ribosomes - rRNA and Proteins mRNA - Nucleotides tRNA – The RNA world theory might explain these three components Aminoacyl-tRNA Synthetase, – A protein, thus a product of translation and cannot be explained away by the RNA world theory L Amino Acids ATP - For energy This appears to be an irreducibly complex system ©2001 Timothy G. Standish ©2001 Timothy G. Standish