Transcript tRNA
From RNA to protein Kanokporn Boonsirichai The coding problem How is the information in a linear sequence of nucleotides in mRNAs translated into the linear sequence of amino acids in proteins? Molecules Monomers Monomer variations DNA Deoxynucleotides 4 kinds RNA Ribonucleotides 4 kinds Protein Amino acids 20 kinds The Genetic Code mRNA sequence is decoded in a set of three nucleotides For a sequence, there exists three possible reading frames. tRNA The code is translated as a triplet of three nucleotides into the corresponding amino acid via a transfer RNA. Codon Anticodon Structure of a tRNA Redundancy of the code More than one tRNAs for some amino acids Some tRNAs can base pair with more than one codon tRNA Biosynthesis Synthesized by RNA polymerase III Synthesized as a larger precursor - trimmed - spliced - base modifications tRNA Splicing Some precursor-tRNAs are with introns tRNAs introns are cut and pasted together by endonuclease and tRNA ligase Cut within the tRNA Paste the tRNA pieces together Coupling of amino acid to tRNAs Achieved by aminoacyl-tRNA synthetases Aminoacyl-t-RNA synthethase How is accuracy achieved? The correct amino acid has the highest affinity for the active site. Hydrolytic editing Nucleotide 1 binding pockets/ acceptor stem recognition mistake in 40,000 tRNA couplings tRNAGln Synthesis of Proteins Formation of a peptide bond between the carboxyl group at the end of a growing polypeptide chain and a free amino group on an incoming amino acid The synthesis is stepwise from the N-terminal end to the C-terminal end. The growing carboxyl end of the polypeptide chain remains activated by its covalent attachment to a tRNA molecule (a peptidyl-tRNA molecule). Peptidyltransferase reaction Decoding mRNAs 1 mistakes in 10,000 amino acid polymerized 2 amino acids are polymerized per second in eukaryotes 20 amino acids are polymerized per second in prokaryotes So, where is it done? What do you on this micrograph? Ribosome Ribosome subunits and their rRNAs are assembled in the nucleolus and exported to the cytoplasm where translation occurs. Translation Elongation A ribosome contains four binding sites for RNA molecules: 3 for tRNAs and 1 for mRNA. tRNA anticodon interacts with the codons on the mRNA in the small subunit of the ribosome. The large subunit catalyzes the peptidyl transferase reaction. A-site: Aminoacyl-tRNA P-site: Peptidyl-tRNA E-site: Exit site Translation Elongation Elongation factors and translation accuracy Ribosomes Contain 2/3 RNAs and 1/3 proteins rRNAs are folded into a highly compact three dimensional structure responsible for the overall shape of the ribosome 23S rRNA forms the catalytic site Translation Initiation Initiator tRNAs always carry the amino acid methionine. The small ribosomal subunit scans the mRNA in the 5’ to 3’ direction . Nucleotides immediately surrounding the start site in eucaryotic mRNAs influence the efficiency of AUG recognition. Bacterial mRNAs are polycistronic Specific sequence for ribosome binding: Shine-Dalgarno sequence Located 8 bp upstream of the start codon. Translation Termination Stop codons (UAA, UGA, UAG) signal the ribosome to stop translation Release factors bind to the ribosome at the A-site causing H2O to be added to the growing peptide chain, releasing it from attachment with the tRNA. eRF1 and tRNA structure comparison Polysomes: multiple ribosomes on a single mRNA Protein synthesis takes 20 seconds to several minutes Ribosomes are spaced ~80 nt apart on an mRNA Translation Accuracy 1 mistake per 10,000 amino acids joined 20 amino acids are incorporated per second in bacteria. Translation is a compromise between accuracy and speed. If too slow, cannot produce enough proteins to survive. Price of Protein Synthesis Four high-energy phosphate bonds must be split per peptide bond. Two to charge the tRNA with an amino acid Two to drive the synthesis itself on the ribosome Extra energy is consumed for incorrectly charged tRNA and for entrance of an incorrect tRNA into the ribosome. Quality control features Both the 5’ cap and the poly-A tail must be recognized before an mRNA enters the ribosome. Why?