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Translation Basics.ppt

  1. Genetic Code, tRNA Translation S.Karthikumar AP/Biotechnology KCET
  2. Genetic Code A G U A C G U A C G U A C G U A C G U A C G U A C G U A C G U A C G U A C G U A C G U A C G U A C G U A C G C C A G C C A G A G A G A G C C G A G C C A U U U U U U U U
  3. Terms  Amino Acid - The building block of proteins.  Genetic Code - The series of codons that make up an organism's DNA  Codon - A three-nucleotide sequence in an mRNA sequence. Used to specify an amino acid.  Reading frame - mRNA sequence yield an amino acid sequence.  Degenerate - Term used to describe the genetic code. Means that more than one codon can specify for a single amino acid.  Stop codon - A codon that is not recognized by a tRNA molecule. One of three codons: UAA, UAG, or UGA. Signals the termination of DNA translation.  Synonyms - Codons that code for the same amino acid.
  4.  Mutation - An error in the genetic code caused most often by a the incorrect substitution, insertion, or deletion of a nucleotide.  Frameshift mutation - One class of genetic code mutation that results from the insertion or deletion of a nucleotide to an mRNA sequence. Results in a new reading frame.  Missense mutation - One class of genetic code mutation that results from the substitution of one base group for another in a codon. Results in the change of the amino acid for which the codon specifies.  Nonsense mutation - One class of genetic code mutation that results from the substitution of one base group for another in a codon. Results in a stop codon, a codon that can no longer be recognized by tRNA molecules.  Silent Mutation - A substitution base mutation that changes a codon, but because of degeneracy does not change the amino acid that codon specifies.  Suppressor mutation - A type of genetic code mutation that alters the result of a different mutation.. It suppresses the effect of first mutation. ( Aminoacid Sequence will be same but nt sequence may vary)  True reversion - One type of suppression mutation that leads to the restoration of the natural genetic code sequence. (Amino acid Sequence as well as nucleotide sequences will be retained.)
  5. The genetic code: how do nucleotides specify 20 amino acids?  4 different nucleotides (A, G, C, U)  Possible codes:  1 letter code  4 AAs <20  2 letter code  4 x 4 = 16 AAs <20  3 letter code  4 x 4 x 4 = 64 AAs >>20  Three letter code with 64 possibilities for 20 amino acids suggests that the  genetic code is degenerate (i.e., more than one codon specifies the same amino acid).
  6. UAG (Amber), UAA (Ochre), UGA (Opal)
  7. Characteristics of the genetic code (written as in mRNA, 5’ to 3’):  Code is triplet. Each 3 nucleotide codon in mRNA specifies 1 amino acid.  Code is comma free. mRNA is read continuously, 3 bases at a time without skipping bases (Some time, translational frameshifting is known to occur).  Code is non-overlapping. Each nucleotide is part of only one codon and is read only once.  Code is almost universal. Most codons have the same meaning in different organisms (Except for mitochondria of mammals).
  8.  Code is degenerate. 18 of 20 amino acids are coded by more than one codon. Met and Trp are the only exceptions.  Code has start and stop signals. AUG codes for Met and is the usual start signal. UAA, UAG, and UGA are stop codons and specify the the end of translation of a polypeptide. UAG (Amber), UAA (Ochre), UGA (Opal)  Wobble occurs in the tRNA anti-codon. 3rd base is less constrained and pairs less specifically.
  9. tRNA
  10. Function Carry amino acids during protein synthesis
  11. tRNA cloverleaf structure  All tRNA molecules are small, ssRNA ranging from 73-93 nts  1st tRNA discovered – tRNAAla  Due to Intra base pairing – cloverleaf structure
  12. Structure 1. The 5'-terminal phosphate group. 2. The acceptor stem is a 7-bp stem made by the base pairing of the 5'-terminal nucleotide with the 3'-terminal nucleotide (which contains the CCA 3'- terminal group used to attach the amino acid). 3. The CCA tail is a CCA sequence at the 3' end of the tRNA molecule. This sequence is important for the recognition of tRNA by enzymes critical in translation.
  13.  4. The D arm is a 4 bp stem ending in a loop that often contains dihydrouridine.  5. The anticodon arm is a 5-bp stem whose loop contains the anticodon.  6. The T arm is a 5 bp stem containing the sequence TΨC where Ψ is a pseudouridine.  7. Bases that have been modified, especially by methylation. The first anticodon base is sometimes modified to inosine (derived from adenine) or pseudouridine (derived from uracil).
  14. Attachment of an amino acid to tRNA molecule  Each aminoacid will attach to specific tRNA by specific aminoacyl synthetase enzyme  Aminoacid is attached with tRNA – called Aminoacylated or Charged tRNA  Example seryl-tRNA or tRNASer  If mischarged; serine is attached with Leu-tRNA - Seryl- tRNALeu  Attachment is by two steps  1. Activation (aa + ATP -> Aminoacyl AMP)  2. Transfer (Aminoacyl AMP + tRNA -> Aminoacyl tRNA + AMP)
  15. Charging of tRNA  charged by attachment of a specific amino acid to their 3’- end to become aminoacyl-tRNA ,  Aminoacylation of aminoacid First, the aminoacyl-tRNA synthetase attaches adenosine monophosphate(AMP) to the – COOH group of the amino acid to creat an aminoacyl adenylate intermediate.  Aminoacyl-tRNA synthetases The synthetase enzymes contact their cognate tRNA by the inside of its L-shaped and use certain parts of the tRNA, called identity elements, to distinguish these similar molecules from one another.
  16. Animation – tRNA charging
  17. Codon-Anti codon Interaction  61 out of 64 possible codons represent 20 aminoacids  It was expected that 61 distinct tRNA molecules  But ~ 40 tRNA molecules available  (Then How 40 tRNA moleculaes recognize 61 codons?)  Only first two bases of codon and anti-codon interact, the 3rd letter doesn’t pair / match  Usually Inosine is present at 3rd position in anticodon  Inosine can pair with either A / U / C  Thus a single tRNA molecule can pair with more than one codons, i.e its synanyms  This concept called – Wobble hyphothesis – proposed by Francis Crick - 1965
  18.  Acceptor site - A three-nucleotide position in a ribosome that binds to an aminoacyl tRNA, a tRNA molecule bearing an amino acid.  Acceptor stem - One secondary structural feature of tRNA. Contains the sequence CCA and has a free 3' –OH. Binds to the amino acid.  Adenylylation - The first step in tRNA charging. Involves the "activation" of an amino acid so that the acid can be bound to a tRNA molecule. The process of activation involves the transfer of an AMP group from ATP to the amino acid.  Aminoacyl tRNA - A tRNA molecule that has been charged. It is loaded with an amino acid and is ready to participate in translation at the ribosome, where it binds to the acceptor site.  Aminoacyl-tRNA synthase - The enzyme that catalyzes the bond between specific tRNA and amino acid, to form aminoacyl tRNA.
  19.  Anticodon arm - A secondary structural feature of tRNA. Contains the anticodon that base pairs with an mRNA codon during translation.  Carboxyl group - A chemical functional group made up of a carbon double- bonded to an oxygen and single-bonded to an –OH group.  Charging - The two-step process in which an amino acid is "loaded" onto a tRNA. The first step is adenylylation; the second is the binding of tRNA and amino acid into an aminoacyl tRNA.  Charged tRNA - Term used to describe a tRNA molecule that has been loaded with an amino acid and is ready to participate in translation.  Dihydrouridine - One of the unusual bases found in tRNA. Contains two additional hydrogens in place of the double bond that is usually found in uracil.  Dihydrouridine arm - A secondary structural feature of tRNA. Contains a number of dihydrouridines.
  20. Ribosomes
  21.  Multicomponent particles involved in protein synthesis  Having proteins and small rRNA (different length)  Both prokaryotes & Eukaryotes are having 1 small sub unit and 1 large sub unit
  22. Eukaryotic cytoplasmic ribosomes are larger and more complex than prokaryotic ribosomes. Mitochondrial and chloroplast ribosomes differ from both examples shown. Ribosome Source Whole Ribosome Small Subunit Large Subunit Prokaryot e 70S 30S 16S RNA 21 proteins 50S 23S & 5S RNAs 31 proteins Eukaryote 80S 40S 18S RNA 33 proteins 60S 28S, 5.8S, &5S RNAs 49 proteins Ribosome Composition (S = sedimentation coefficient)
  23. Prokaryotic Ribosomes •50 s and 30 s subunits association and dissociation is • based on the conc. Of MgCl2. •At high Con. – Association •At Low Conc. – Dissociation Small Sub Unit – Binding of mRNA large sub Unit – Binding of tRNA
  24. Translation - Direction Movement of Ribosome on mRNA is 5’ to 3’ end Synthesis of protein peptide from Amino terminal to Carboxyl terminal
  25. Translation….