Ch 13 Translation and Proteins Translation of mRNA Depends on Ribosomes and Transfer RNAs o genetic code stores information as triplet nucleotides in DNA information transcribed into a mRNA that is complementary to one DNA strand o final gene product of gene expression linear series of amino acids as prescribed by the genetic code o Translation of mRNA biological polymerization of amino acids into polypeptide chains o This process requires amino acids messenger RNA mRNA ribosomes transfer RNA tRNA o Occurs only in association with ribosome s o mRNA runs through the ribosome and amino acid is polymerized into o tRNAs adapt genetic information present as specific triplet codons in mRNA to their corresponding amino acid polypeptide o tRNAs have anticodons that complement the mRNAs base pairs with codon carry the corresponding amino acid H bonding of tRNAs to mRNA hold amino acid in proximity so peptide bond can be formed Ribosomal structure o Ribosomes ribosomal proteins and ribosomal RNAs rRNAs have a large subunit and a small subunit called monosomes when they join together o The main differences between prokaryotic and eukaryotic ribosomes are summarized o rRNAs perform important catalytic functions associated with translation promote the binding of the various molecules involved in translation and fine tune the process o rRNA genes rDNA code for rRNA part of a moderately repetitive DNA fraction present in clusters at various chromosomal sites Each cluster in eukaryotes contains tandem repeats separated by a noncoding spacer DNA sequence tRNA structure o tRNAs small in size and are very stable o Composed of 75 90 nucleotides o Identical structures in prokaryotes eukaryotes o Transcribed from DNA and contain posttranscriptionally modified bases enhance hydrogen bonding efficiency during translation o I can bind to U A C o two dimensional structure of tRNAs cloverleaf due to base pairing o series of and loops paired stems unpaired consistently modified o tRNA has an anticodon complementarily base pairs with the codon o At the other end is the 3 acceptor region where the amino acid is in the mRNA bound Charging tRNA o Before translation can proceed tRNA molecules must be chemically linked to their respective amino acids This activation process charging or aminoacylation is done by aminoacyl tRNA o There are 20 different synthetases one for each amino synthetase acid highly specific recognize only one amino acid o Due to the ability of the third member of the triplet code to wobble 31 tRNAs rather than 61 for the 61 triplets that encode amino acids o Covalent linkage formaed between 5 phosphate group of ATP and carbonyl end of amino acid o Remains associated with the enzyme complex that then reacts with specific tRNA molecule amino acid then transferred to appropriate tRNA and binds covalently to 3 end Translation of mRNA can be divided into 3 steps o Translation is divided into three phases initiation elongation and termination o Many of the protein factors and their roles in translation are summarized o Initiation requires the small and large ribosomal subunits mRNA GTP Mg2 charged initiator tRNA initiation factors IFs enhance binding affinity of translational components Shine Dalgarno sequence prok o Purine rich sequence upstream of start codon binds to small subunit of ribosome o base pairs with a region 16S rRNA of the 30S small subunit facilitating initiation Kozak sequence euk o ACCAUGG similar function Another initiation protein enhances the binding of charged fmet tRNA to the small subunit in response to the AUG triplet initiation complex then combines with the large ribosomal subunit initiation factors o Molecule of GTP hydrolyzed energy for release of Elongation requires both ribosomal subunits assembled with the mRNA P peptidyl site and A aminoacyl site o The lengthening of the growing polypeptide chain by one amino acid elongation o Charged tRNA binds to Psite when AUG triplet of mRNA is in correct position of small subunit o 2nd triplet in mRNA dictates which charged tRNA molecule will go to Asite small subunit decodes mRNA large subunit catalyzes peptide bond synthesis P site peptidyl site E site exit site E site tRNA uncharged A site aminoacyl site o charged tRNAs enter the A site peptidyl transferase catalyzes peptide bond covalent formation between the amino acid on the tRNA at the A site and the growing peptide chain bound to the tRNA in the P site by distance of 3 nucleotides o uncharged tRNA moves to the E exit site o tRNA bound to the peptide chain moves to the P site o sequence of elongation and translocation is repeated over and over o elongation process requires several elongation factors as well as energy derived from the hydrolysis of GTP 3rd triplet of mRNA now in position to accept another specific charged tRNA into A site o Once an amino acid chain of reasonable size assembled about 30 amino acids emerges through a tunnel in the base of the large subunit o small subunit decodes the triplets present in mRNA and the large subunit is responsible for peptide bond synthesis Termination o signaled by stop codon termination codon nonsense codon UAG UAA UGA A site for a tRNA in the A site These codons do not specify an amino acid nor do they call o GTP dependent release factors cleave the polypeptide chain from the tRNA and release it from the translation complex o tRNA released from the ribosome disassociates into its o finished polypeptide still attached to P site and A site is empty subunits o Stop codons ORF UGA UAA UAG no amino acids for these codons part of o Polyribosomes o Polysomes or polyribosomes mRNAs with several ribosomes translating at once As elongation proceeds initial portion of mRNA passed through ribosome mRNA free to associate with another small unit second initiation complex polyribosome High Resolution Studies Have Revealed Many Details about the Functional Prokaryotic Ribosome o crystal structures of the individual ribosomal subunits and the intact 70S bacterial ribosome with associated mRNA and tRNAs have been o entire translational complex size and shape was seen at the atomic o These studies show visualized level that the shape of the ribosomes changes during different functional states catalytic steps that join together amino acids during translation occur under the direction of RNA o All three sites A P and E have been identified in X ray diffraction studies o energetic requirement for achieving the various conformational changes
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