* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Nucleoside Phosphoramidate Monoesters: Potential
Fatty acid metabolism wikipedia , lookup
Fatty acid synthesis wikipedia , lookup
Eukaryotic transcription wikipedia , lookup
RNA silencing wikipedia , lookup
RNA polymerase II holoenzyme wikipedia , lookup
Ribosomally synthesized and post-translationally modified peptides wikipedia , lookup
Transcriptional regulation wikipedia , lookup
Two-hybrid screening wikipedia , lookup
Silencer (genetics) wikipedia , lookup
Polyadenylation wikipedia , lookup
Proteolysis wikipedia , lookup
Peptide synthesis wikipedia , lookup
Metalloprotein wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Messenger RNA wikipedia , lookup
Point mutation wikipedia , lookup
Gene expression wikipedia , lookup
Protein structure prediction wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Amino acid synthesis wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Biochemistry wikipedia , lookup
Genetic code wikipedia , lookup
Epitranscriptome wikipedia , lookup
Formation of RNA Polymerase II pre-initiation complex IID contains TBP that binds TATA box IIA stabilizes IID binding to promoter IIB binds initiation sequence Pol II binds IIB IIE stimulates transcription IIH has kinase and helicase activity RNA Synthesis in E. Coli Transcription bubble RNA splicing in eukaryotes Primary transcript, hnRNA Alternative splicing patterns give rise to multiple proteins from the same pre-mRNA RNA Synthesis: Take Home Message 1) DNA sequences are translated into RNA messages by RNA polymerases. 2) The initiation of RNA synthesis is controlled by specific DNA promoter sequences. 3) The synthesis of RNA is governed by initiation, elongation, and termination steps. 4) Eukaryotic mRNA is extensively processed Overview of Protein Synthesis (Translation). Transfer RNA. Required reading: Stryer’ Biochemistry 5th edition Ch. 5, p. 132-136, Ch. 28 p. 797, Ch. 29 p. 813-823 or Stryer 4th edition p. 102-104, 109-112, Ch. 34, p. 875-888 and Ch. 33 p. 849-850 Flow of Genetic Information replication DNA RNA Proteins transcription translation DNA Cellular Action How do we go from mRNA to Protein? DNA mRNA Protein t-RNA Amino Acid Sequence Transfer RNA • Acts as an adaptor molecule between mRNA and peptide sequence • Contains amino acid attachment site and template recognition site NH2 N tRNA N O oO N P O N O H H H H O O OH C CH NH3 R aminoacyl tRNA synthetase Translation of mRNA 5’ N 3’ C • mRNA is read sequentially from a fixed starting point • Sequence of 3 Bases = One Codon (no gaps) • One Codon = One Amino Acid • 20 Natural Amino Acids = 64 Codons (43) “Degenerate Code: Several Different Codons per Amino Acid” Genetic Code During translation, mRNA passes through the ribosome so that each codon recognizes its tRNA Translating the Message DNA 5'-ATG-GCC-TTT-GAT-TCT-AAA-TAA-3' RNA 5'-AUG-GCC-UUU-GAU-UCU-AAA-UAA-3' Protein N- met ala phe asp ser Correct reading frame is essential lys stop -C Write the sequence of amino acids in a polypeptide translated from the following mRNA: 5’ GGA GGA GUA AGU UGU Gly– Gly – Val – Ser - Cys The genetic code is nearly universal, with the exception of mitochondria Transfer RNA Secondary Structure of Transfer RNA molecule 60-93 nt long 7 bp acceptor stem O O H2C H2C NH NH N O dihydrouridine (UH2) HN O pseudouridine ( Base sequence of yeast tRNAAla: cloverleaf folding result from the presence of “self complementary” regions -UCCGGTCGAUUCCGGA- tRNA has an “L” Tertiary Structure T Loop Acceptor Stem D Loop V Loop Anti-Codon Amino acid attachment site Tertiary base pairs are responsible for the tertiary structure of tRNA #46 (m7G) #22 G Tertiary base pairs in tRNAPhe #13 C #46 (m7G) #22 G #13 C Tertiary base pairs in tRNAPhe Non-standard H bond interactions, some linking 3 bases, help stabilize the L-shaped tertiary structure of tRNA. tRNA Formation in E. Coli • 60 genes for tRNA are clustered in 25 transcription units • tRNA Precursors containing several tRNAs are cleaved with RNases: RNase P RNase P 5' 3' CCA CCA RNase D RNase D RNase P – generates 5’ ends of tRNA by cleaving the bond 5’ to each tRNA RNase D – trims the 3’ end up to the CCA sequence tRNA Activation (charging) by aminoacyl tRNA synthetases Aminoacyl tRNA synthetase Two important functions: 1. Implement genetic code 2. Activate amino acids for peptide bond formation The key enzymes: Amanoacyl-tRNA synthetases Aminoacyl-tRNA Synthesis Summary of 2-step reaction: 1. amino acid + ATP aminoacyl-AMP + PPi 2. aminoacyl-AMP + tRNA aminoacyl-tRNA + AMP The 2-step reaction is spontaneous overall, because concentration of PPi is kept low by its hydrolysis, catalyzed by Pyrophosphatase. tRNA Activation by aminoacyl tRNA synthetases 1. Aminoacyl-AMP formation: HO O (-)O O P R O O(-) O P C O O +H 3N O O(-) +H 3N R P O- Adenine O O C O O Adenine O P O O- + PPi OH OH Aminoacyl adenylate (Aminoacyl-AMP) OH OH 2Pi 2. Aminoacyl transfer to the appropriate tRNA: R R O +H 3N C O O P O O- Adenine O + HO-ACC-tRNA O +H 3N C ACC-tRNA + AMP O OH OH Overall reaction: amino acid + tRNA + ATP aminoacyl-tRNA + AMP + PPi Classes of Aminoacyl-tRNA Synthetases • Class I: Arg, Cys, Gln, Glu, Ile, Leu, Met, Trp, Tyr, Val (Generally the Larger Amino Acids) • Class II: Ala, Asn, Asp, Gly, His , Lys, Phe, Ser, Pro, Thr (Generally the smaller amino acids) Main Differences between the two classes: 1. Structural differences. Class I are mostly monomeric, class II are dimeric. 2. Bind to different faces of the tRNA molecule 3. While class I acylate the 2’ hydroxyl of the terminal Ado, class II synthetases acylate the 3’-OH Class I and II synthetases bind to different faces of the tRNA molecule Class I synthetases acylate the 2’-OH Class II synthetases acylate the 3’-OH NH2 NH2 tRNA N tRNA N N N O O oo- O N N P O N P O O O O H H H H H H OH O O H O H O OH C C CH CH R NH3 R NH3 N The accuracy of protein synthesis depends on correct charging of tRNAs with amino acids 1. tRNA synthetases must link tRNAs with their correct amino acids. 2. tRNA synthetases recognize correct amino acids by specific binding to the active site and proofreading. 3. tRNA synthetases recognize correct tRNAs via by interacting with specific regions of tRNA sequence. The accuracy of protein synthesis depends on correct charging of tRNAs with amino acids 1. tRNA synthetases must link tRNAs with their correct amino acids. 2. tRNA synthetases recognize correct amino acids by specific binding to the active site and proofreading. 3. tRNA synthetases recognize correct tRNAs via by specific regions of tRNA sequence. The acylation site of threonyl tRNA synthetase contains a Zinc ion that interacts with the OH group of Threonine O H 2N CH C O OH H 2N CH C CH OH CH CH3 CH3 CH3 Thr Val OH Threonyl-tRNA synthetase contains editing site O H 2N CH C CH OH O OH H 2N CH C H 2C CH3 Thr Ser OH OH tRNA Synthetase Proofreading •“Double sieve” based on size • Flexibility of the acceptor stem essential tRNA Synthetase Proofreading Larger Acylation Site Larger Acylation Site Smaller Hydrolytic Site Smaller Hydrolytic Site CH 3 H 3C CH 3 CH 3 O O NH 3+ +H 3N tRNAIle O CH 3 O tRNAIle Difference in Size H 3C CH 3 O O +H 3N +H 3N O CH 3 O tRNAIle Ile Correct Acylation Val Misacylation tRNAIle tRNAVal Synthetase Proofreading: hydrophobic/polar recognition motif Hydrophobic Acylation Site 3HC Polar Hydrolytic Site Hydrophobic Acylation Site Polar Hydrolytic Site CH 3 H 3C O OH O +H 3N NH 3+ O tRNAVal tRNAVal O Difference in Hydrophobicity CH3 CH 3 HO CH 3 O O +H 3N +H 3N O tRNAVal Val Correct Acylation O tRNAVal Thr Misacylation The accuracy of protein synthesis depends on correct charging of tRNAs with amino acids 1. tRNA synthetases must link tRNAs with their correct amino acids. 2. tRNA synthetases recognize correct amino acids by specific binding to the active site and proofreading. 3. tRNA synthetases recognize correct tRNAs via using specific regions of the tRNA sequence. tRNA Recognition by Synthetases • different recognition motif depending on synthetase • usually just a few bases are involved in recognition •Can involve specific recognition of the anticodon (e.g. tRNAMet), stem sequences can (e.g. tRNAAla), both stem regions and anticodon (e.g. tRNAGln), or, less frequently, D loop or T loop bases. Examples of tRNA Recognition by aminoacyl tRNA Synthetases tRNAAla 5'P G3 3'OH A C C tRNAPhe 5'P tRNASer 3'OH A C C 5'P U70 C11 A G24 D G34 A36 A35 3'OH A C C Threonyl tRNA synthase complex with tRNA Codon-anticodon recognition between tRNA and mRNA The relationship between the number of codons, tRNAs, and synthetases Total of 61 codons, but not 61 tRNAs! The same tRNA can recognize more than one codon Example: Codon tRNA GCU GCC GCA tRNAAla (5’-IGC-3’) alanyl tRNA synthetase 3’ Synthetase 5’ CGI 5’-GCU (C,A)-3’ anticodon codon Codon : Anticodon Recognition 1. The first two interactions (XY-X’Y’) obey Watson-Crick base pairing rules. 2. The third interaction is less strict (Wobble pairing is allowed) 3 2 1 t RNA- 3'-X Y Z -5' anticodon mRNA- 5'-X’Y’Z’-3' codon 1 2 3 The Third Base of Codon is Variable Wobble base pairing rules 5’ anticodon base 3’ codon base C G A U U A or G G C or U I U, C, or A tRNA Anticodon-Codon Recognition Adenosine Guanosine Inosine NH 2 O O N N N HN N H N N N N HN HN N H N Ribose Anticodon Codon 3' 5' C G NH2 O O I C N C-I base pair 5' 3' 3' 5' C G NH2 G C N C1' C1' N 5' 3' 3' 5' N A-I base pair C G G C 5' 3' I U O N N HN HN I A O N N N HN N C1' G C NH N N C1' C1' O N O HN N N U-I base pair C1' tRNA Anticodon-Codon Recognition Anticodon Codon 3' 5' C G G C 5' 3' I U 3' 5' C G Anticodon Codon 3' 5' C G G C G U 5' Anticodon Codon 3' 5' C G G C U A 5' Anticodon Codon 3' 5' C G G C C G 5' 3' 3' 3' G C I C 5' 3' 3' 5' C G G C G C 5' 3' 5' C G G C U G 5' 3' 5' C G G C A U 5' 3' 5' C G G C 3' 3' 3' I A 5' 3' Genetic Code Nonsense suppression Nonsense mutations = change of codon for an aa to STOP Usually lethal – truncated protein Can be rescued by mutation in a different part of the genome Mechanism: tRNA gene mutation Example: E. Coli Amber suppressor tRNATyr anticodon change GUA CUA Mutated tRNA recognized stop codon as Tyr and prevents chain termination Overview of Protein Synthesis : Take Home Message 1) Translation of the genetic code is dependent on three base words that correspond to a single amino acid. 2) The mRNA message is read by tRNA through the use of a three base complement to the three base word. 3) A specific amino acid is conjugated to a specific tRNA (three base word). 4) Amino acid side chain size, hydrophobicity and polarity govern the ability of tRNA synthetases to conjugate a specific three base message with a specific amino acid.