Week 5 Textbook Notes

BIO 130 – Week 5 Pages 366-390 From RNA to Protein An mRNA Sequence is Decoded in Sets of Three Nucleotides • The conversion of the information in RNA into protein is called translation • The sequence of nucleotides in the mRNA molecule is read in consecutive groups of three o Each group of three consecutive nucleotides in RNA is called a codon, and each codon specifies either one amino acid or a stop to the translation process tRNA Molecules Match Amino Acids to Codons in mRNA • The codons in an mRNA molecule do not directly recognize the amino acids they specify: the group of three nucleotides does not, for example, bind directly to the amino acid • The translation of mRNA into protein depends on adaptor molecules that can recognize and bind both to the codon and, at another site on their surface, to the amino acid • These adaptors consist of a set of small RNA molecules known as transfer RNAs (tRNAs) • tRNA has two regions of unpaired nucleotides situated at either end of the L-shaped molecule o One of these regions forms the anticodon, a set of three consecutive nucleotides that pairs with the complementary codon in an mRNA molecule o The other is a short single-stranded region at the 3’ end of the molecule: this is the site where the amino acid that matches the codon is attached to the tRNA • Some amino acids have more than one tRNA and some tRNAs are constructed so that they require accurate base-pairing only at the first two positions of the codon and can tolerate a mismatch at the third position • Aminoacyl-tRNA synthetases covalently couples each amino acid to its appropriate set of tRNA molecules Editing by tRNA Synthetases Ensures Accuracy • The synthetase must first select the correct amino acids, and most synthetases do so by a two step mechanism: o (1) The correct amino acid has the highest affinity for the active-site pockets of its synthetase, and is thus favoured over the other 19 o (2) When tRNA binds the synthetase, it tries to force the amino acid into a second pocket in the synthetase, the precise dimensions of which exclude the correct amino acid but allow access by closely related amino acids BIO 130 – Week 5  If an amino acid enters this editing pocket, it is hydrolyzed from the AMP and it released from the enzyme The RNA Message is Decoded in Ribosomes • Protein synthesis is performed in the ribosome, a complex catalytic machine made from more than 50 different proteins and several RNA molecules, the ribosomal RNAs (rRNAs) • Eukaryotic ribosomes subunits are assembled at the nucleolus, when newly transcribed and modified rRNAs associate with ribosomal proteins, which have been transported into the nucleus after their synthesis in the cytoplasm o The two ribosomal subunits are then exported to the cytoplasm, where they join together to synthesize proteins • Ribosomes consist of one large and one small subunit • The small subunit provides the framework on which the tRNAs can be accurately matched to the codons of the mRNA, while the large subunit catalyzes the formation of the peptide bonds that link the amino acids together into a polypeptide chain o When not actively synthesizing proteins, the two subunits of the ribosome are separate and join together on an mRNA molecule, usually near its 5’ end, to initiate the synthesis of protein • A ribosome contains four binding sites for RNA molecules: one is for the mRNA and three, (called the A-site, the P-site, and the E-site) are for tRNAs • Once protein synthesis has been initiated, each new amino acid is added to the elongating chain in a cycle of reactions containing four major steps: tRNA binding, peptide bond formation, large subunit and small subunit translocation o (1) A tRNA carrying the next amino acid in the chain binds to the ribosomal A-site by forming base pairs with the mRNA codon positioned there, so that the P-site and the A-site contain adjacent bond tRNAs o (2) The carboxyl end of the polypeptide chain is released from the tRNA at the P- site and joined to the free amino group of the amino acid linked to the tRNA at the A-site, forming a new peptide bond – this reaction is catalyzed by a peptidyl transferase contained in the large ribosomal subunit o (3) The large subunit moves relative to the mRNA held by the small subunit thereby shifting the acceptor stems of the two tRNAs to the E- and P-sites of the large subunit o (4) Another series of conformation changes moves the small subunit and its bound mRNA exactly three nucleotides, resetting the ribosome so it is ready to receive the next aminoacyl-tRNA Elongation Factors Drive Translation Forward and Improves Its Accuracy BIO 130 – Week 5 • Two elongation factors enter and leave the ribosome during each cycle, each hydrolyzing GTP to GDP and undergoing conformational changes in the process o These factors are called EF-Tu & EF-G in bacteria and EF1 & EF2 in eukaryotes o Without these elongation factors this process is very slow, inefficient, and inaccurate • EF-Tu and EF-1 increases the accuracy of translation: o (1) As it escorts an incoming aminoacyl-tRNA to the ribosome, EF-Tu checks whether the tRNA-amino acid match it correct o (2) EF-Tu monitors the initial interaction between the anticodon of an incoming aminacyl-tRNA and the codon of the mRNA in the A-site  Aminoacyl-tRNAs are bent when bound to the GDP-form of EF-Tu, this bent configuration allows codon pairing but prevents incorporation of the amino acid into the growing polypeptide chain  If the codon-anticodon match is correct, the ribosome rapidly triggers the hydrolysis of the GTP molecule, whereupon EF-Tu releases its grip on the tRNA and dissociates from the ribosome, allowing the tRNA to donate its amino acid for protein synthesis  The codon-anticodon interactions are gauged by the small subunit of the ribosome. rRNA in the ribosome subunit forms a series of hydrogen bonds with the codon-anticodon pair that allows determination of its correctness • Another proofreading mechanism: o Incorrectly matched tRNAs dissociate more rapidly than those correctly bound, therefore most incorrectly bound tRNA molecules will leave the ribosome without contributing to the polypeptide chain • RNA molecul