Translation - Final (Class) (2) PDF

BS207 –CELL BIOLOGY GENE EXPRESSION II: TRANSLATION  Proteins Perform Many Diverse Roles Hemoglobin and Myoglobin Transport oxygen, which is essential Immunoglobulins (Antibodies) Function in immune system of vertebrates for cellular meta...

BS207 –CELL BIOLOGY GENE EXPRESSION II: TRANSLATION  Proteins Perform Many Diverse Roles Hemoglobin and Myoglobin Transport oxygen, which is essential Immunoglobulins (Antibodies) Function in immune system of vertebrates for cellular metabolism Transport proteins Movement of molecules across membranes Collagen and Keratin Structural proteins associated with Hormones and their Receptors skin, connective tissue, and hair of Regulate various types of chemical organisms activity Histones Actin and Myosin Bind to D N A in eukaryotes Contractile proteins found in muscle tissue Transcription factors Regulate gene expression Tubulin Enzymes Basis of microtubule function in Most diverse and extensive group of mitotic and meiotic spindle fibers proteins  Amino acids 20 amino acids commonly found in proteins (see fig.) Each amino acid has two abbreviations in universal use; for example, alanine is designated either Ala/A Modified amino acids: Some of the 20 amino acids may be modified giving rise to new amino acids e.g., lysine to pyrrolysine Amino acids Central carbon atom bonded to Carboxyl group, Amino group, H & R (radical) group R group (or side chain) can vary in property: Nonpolar (hydrophobic) or Polar (hydrophilic) or Positively and negatively charged Peptide bond and Polar nature of Peptides Dehydration (condensation) reaction facilitates peptide bond formation between two amino acids Reaction occurs between carboxyl group of one amino acid and amino group of another All peptide/polypeptide chains have a N-terminus at one end and a C-terminus at the other.  Genetic Codons: Triplets of Nucleotides The flow of information from gene to protein is based on a 3-nucleotide genetic codons or triplet code: Reading Frame: Codons must be read in the correct reading frame (correct groupings) in order for the specified polypeptide to be produced Each codon (3-nucleotide code) specifies an amino acid. Codons are non-overlapping and translated into a chain of amino acids, forming a polypeptide Evolution: The genetic code is nearly universal, shared by the simplest bacteria to the most complex animals Genes can be transcribed and translated after being transplanted from one species to another  Number of Genetic Codons 64 Genetic Codons 61 amino acid codons - for the 20 amino acids Includes one Start codon - all organisms have “AUG” start codon that codes for amino acid Met (methionine) 3 Stop Codons - code for “stop” signals to end translation: UAA, UAG, UGA Stop codons do not code for amino acid Genetic Codons are Redundant, Genetic code is described as degenerate, or redundant, because a single amino acid may be specified by more than one codon. Each codon specifies only one amino acid  Point or Base Substitution Mutations Point Mutation /Base Substitution Mutation - Single base change in codon e.g. CCA > CCU Three types of point mutations 1. Silent mutation: amino acid specified remains same before and after mutation (because of redundancy in the genetic code) 2. Missense mutation: amino acid specified is different after mutation 3. Nonsense mutation : changes amino acid specifying codon into a stop codon; nearly always leading to a nonfunctional protein  InDel Mutations & Frameshift Base insertion or deletion may produce frameshift mutation. Frameshift mutations alter the reading frame of codons InDel mutations: Insertion is the addition of nucleotide while Deletion is loss of nucleotide pairs in a gene InDel mutations may change the entire reading frame and amino acid sequence from the point of the mutation. Hence they have a disastrous effect on the resulting protein.  Translation Requirements Translation Is the translation of a unique sequence of mRNA codons to a unique sequence of amino acids polymerized into polypeptide chains Is the translation of nucleic acid language to protein language Translation Requirements Amino acids Messenger R N A (mRNA) Transfer R N A (tRNA) Ribosomes Enzymes Translation Requirements Initiation Elongation Termination  Transfer RNA (tRNA) tRNA —transfer R N A Transcribed from DNA, small in size (75–90 nucleotides) and very stable Contain posttranscriptionally modified bases Help in the translation process Bring amino acid specified by a mRNA codon to its appropriate location tRNA has a cloverleaf structure Cloverleaf structure includes: Anticodon stem, Amino acid acceptor stem, T stem & D stem The single stranded tRNA folds due to intra-strand complementarity held in place by H-bonds tRNA Anticodon Each tRNA anticodon is complementary to a specific mRNA codon Codon-Anticodon pairing is complimentary and antiparallel tRNA Aminoacid Amino acid is covalently linked to the CCA sequence at the 3′ end of all tRNAs Each tRNA carries a specific amino acid.  Aminoacylation: tRNA Charging Aminoacylation: Each tRNA carries a specific amino acid. Amino acid is chemically linked to tRNA by a process called Aminoacylation. Enzyme Aminoacyl tRNA synthetase catalyzes aminoacylation to 3’ end of tRNA Amino acid is covalently linked to the CCA sequence at the 3′ end of all tRNAs Synthetases are highly specific; recognize only one amino acid  Wobble Hypothesis The Wobble hypothesis The initial two ribonucleotides of mRNA codons are more critical than the third Third mRNA codon base Is less spatially constrained Need not adhere as strictly to established base-pairing rules Forms loose wobble pair with 1st tRNA anticodon base Inosine (I) is a modified tRNA nitrogenous base that can pair with A, C or U to form the wobbly pair  Ribosomes Have an essential role in expression of genetic information Ribosome Composition Consist of ribosomal proteins and ribosomal RNA (rRNA) Consists of large and small subunits Prokaryote ribosomes are 70S (50S + 30S) Eukaryote ribosomes are 80S (60S + 40S) Ribosome Structure A site: tRNA-amino acid arrival site P site: peptide bond formation site E site: tRNA exit site Codons and tRNAs move from A -> P - >E as translation progresses  Initiation of Translation -Prokaryotes Translation occurs in the 5’ 3’ direction Initiation in Bacteria 1) Initiation Factor 1 (IF1) binds to 30S subunit and prevents premature entry of tRNA to A site 2) IF3 binds to 30S subunit and prevents premature binding of 50S subunit and facilitates mRNA binding 3) mRNA bind to 30S with the help of Shine-Dalgarno sequence - AGGAGGU Shine Dalgarno sequence in mRNA is recognized by a complimentary sequence in16S rRNA in 30S small subunit - facilitating initiation Shine-Dalgarno precedes AUG start codon in bacteria  Initiation of Translation - Prokaryotes 4) IF2-GTP helps in binding of fMet-tRNA to 30S subunit 5) The 50S subunit now combines with the 30S subnunit 6) With this the Initiation complex is complete 7) All initiation factors –IF1-3, GDP are released 50S Initiation of Translation - Eukaryotes 1) Initiation Complex The process is same as prokaryotes Eukaryotic initiation factors – eIF1, eIF2, eIF3 help in forming initiation complex 2) Kozak sequence of mRNA mRNA Kozak sequence binds to 18S rRNA in 40S small subunit of eukaryotic ribosome Met- tRNA binds to AUG codon at P site with the help of eIF2 and GTP The 60S subunit now combines with the 40S subunit With this the Initiation complex is ready  Elongation of Translation Steps are same in pro- and eukaryotes 1 Translation continues in the 5’  3’ direction. Several Elongation factors are involved in both pro- and eukaryotes 1) Fig 1: Start AUG codon positions at P site (not A site) Initiator fMet-tRNA (N-Formylmethionine-tRNA) enters P site in bacteria; For eukaryotes it is – Met-tRNA MetRNA is the only tRNA that does not to enter A site. Instead positions at P site 2 23S rRNA (prokaryotes) & 28S rRNA (eukaryotes) in the large subunits are ribozymes Also known as Peptidyl transferase - catalyze peptide bond formation between amino acids and transfer the elongating chain to incoming tRNA as it shifts to P site 2) Fig. 2 3 2nd codon is read at A site Phe-tRNA latches on to 2nd codon Peptide bond forms between Met-Phe amino acids Elongating peptide chain Met-Phe shifts to tRNA in A site 3) Fig. 3 Codons & tRNAs in P and A sites shift one place to left As the shift occurs the elongating peptide chain is translocated to 4 the tRNA shifting to P site tRNA in P site moves to E site & exits ribosome 4) Fig. 4  Termination of Translation Termination Signaled by stop codons (U A G, U A A, U G A) in A site Codons do not specify any amino acid GTP-dependent release factors Stimulates hydrolysis of polypeptide from peptidyl tRNA—released from translation complex Summary – Translation Process  Polyribosomes Polysomes (or Polyribosomes) Presence of polyribosomes indicates active protein synthesis mRNAs with several ribosomes translating at once As mRNA passes through ribosome, its free to associate with another small subunit  Posttranslational Modifications Posttranslational modifications Modifications are crucial to functional capability of final protein product N-terminus amino acid removed or modified Individual amino acid residues modified Carbohydrate side chains are sometimes attached Polypeptide chains may be trimmed Polypeptide chains often complexed with metals