©2001 Timothy G. Standish Proverbs 3:1-3 1My Son, forget not my law; but let thine heart keep my commandments: 2For length of days, and long life, and peace, shall they add to thee. 3Let 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.

mRNA Transcription Introduction The Central Dogma of Molecular Biology Cell Polypeptide (protein) Translation Ribosome Reverse transcription DNA

©2001 Timothy G. Standish 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 5’ 3’ 5’ Template (antisense) strand Coding (sense) strand

©2001 Timothy G. Standish Transcription 5’ 3’ 5’ Template (antisense) strand Coding (sense) strand 5’ RNA Pol.

©2001 Timothy G. Standish Transcription 5’ 3’ 5’ RNA Pol. Template (antisense) strand Coding (sense) strand

©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: 1Ribosomal RNA (rRNA) - Several rRNAs are vital constituents of ribosomes 2Transfer RNA (tRNA) - The molecule that physically couples nucleic acid codons with specific amino acids 3Messenger 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 Transcription can be logically divided into three distinct stages: 1. Initiation 2. Elongation 3. Termination

©2001 Timothy G. Standish A “Simple” Gene Protein Coding Region Terminator Sequence Promoter/ Control Region Transcription Start Site 3’ Untranslated Region 5’ Untranslated Region 3’5’ RNA Transcript

©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 RNA Pol.Initiation T. F. RNA Pol. 5’ RNA Promoter T. F.

©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 RNA Pol. 5’ RNA Pol. 5’ RNA Pol. 5’ RNA Termination Rho Independent Terminator

©2001 Timothy G. Standish RNA Pol. 5’ RNA Termination Rho Dependent Terminator  RNA Pol. 5’ RNA  Pol. 5’ RNA Help, Rho hit me! 

©2001 Timothy G. Standish Differences Between Transcription In Prokaryotes and Eukaryotes

©2001 Timothy G. Standish 3’ 5’ 3’ Transcription And Translation In Prokaryotes Ribosome 5’ mRNA RNA Pol.

©2001 Timothy G. Standish DNA Cytoplasm Nucleus Eukaryotic Transcription Export G AAAAAA RNA Transcription Nuclear pores G AAAAAA RNA Processing mRNA

©2001 Timothy G. Standish A “Simple” Eukaryotic Gene Terminator Sequence Promoter/ Control Region Transcription Start Site RNA Transcript 5’ Untranslated Region 3’ Untranslated Region Exons Introns 3’5’ Exon 2Exon 3 Int. 2 Exon 1 Int. 1 3’5’ Exon 2Exon 3Exon 1 Int. 2Int. 1

©2001 Timothy G. Standish 3’5’ Exon 2Exon 3 Int. 2 Exon 1 Int. 1 Processing Eukaryotic mRNA Protein Coding Region 3’ Untranslated Region5’ Untranslated Region 3’ AAAAA 3’ Poly A Tail 5’ G 5’ Cap Exon 2 Exon 3Exon 1 Int. 2Int. 1 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 HH C O C N C O N C C C C C H HO H OP O HOH O 5’ End of hnRNA H H C O C N C O N C C C C C H H O H H O P O H O O P O H H O O P O O H O GTP

©2001 Timothy G. Standish The 5’ Cap HH C O C N C O N C C C C C H HOH H OP O H O P O O HO O P O H H O O H HH C O C N C O N C C C C C H HOH H OP O HO O

©2001 Timothy G. Standish Transfer RNA (tRNA) Acts as the adapter molecule between the genetic code on mRNA and the protein “language” 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 D Arm - Contains dihydrouridine Acceptor Arm - A specific amino acid is attached to the 3’ end T  C arm -  stands for pseudouridine Extra Arm - May vary in size Anticodon Transfer RNA (tRNA) U*U* Pu Py :1 G*G* 17:1 Pu A 20: G A G C Pu 59  A*A* C Py T Pu * U Py * :1 47:15 46 Py 47: C C A Amino Acid attachment site

©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

Gly Amino- acyl-tRNA Synthetase Gly Amino- acyl-tRNA Synthetase A P Making Aminoacyl- tRNA P P Pyrophosphate A P P P ATP Amino- acyl-tRNA Synthetase A P Gly CCA

Amino- acyl-tRNA Synthetase Making Aminoacyl- tRNA Gly CCA Aminoacyl- tRNA 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 Gly P P Pyrophosphate A P P P ATP Amino- acyl-tRNA Synthetase Gly Amino- acyl-tRNA Synthetase A P A P AMP Amino- acyl-tRNA Synthetase Gly CCA

©2001 Timothy G. Standish Aminoacylation of tRNA H H C O C N C O N C C C C C HHO H H O P O H O N N C C O HH H N H H 3’ 5’ H CN C O H R H HO H OH

©2001 Timothy G. Standish Amino acid tRNA Aminoacylation of tRNA 3’ 5’ H H C O C N C O N C C C C C H HO H H O P O H O N N C C O H H N H H H CN C O H R H H OH Class I Aminoacyl tRNA Synthetases attach amino acids to the 2’ carbon while Class II attach to the 3’carbon

©2001 Timothy G. Standish Classification of Aminoacyl- tRNA Synthetases Class I - 2’ OH Glu (  ) Gln (  ) Arg (  ) Val (  ) Ile (  ) Leu (  ) Met (    Tyr (      (     Class II - 3’ OH Gly (    2  Ala (  4  Pro (    Ser (    Thr (    Asp (   ?? Asn (    His (    Lys (    Aminoacyl-tRNA Synthetases (ARS) may be mono or multimeric. Two types of polypeptide chains are recognized:  and .

©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