BMB 251 1st Edition Lecture 21 Outline of Last Lecture I. ClickersII. Splicing and Polyadenylation via C-terminal tailIII. RNA polymerase I, II, and IIIIV. Nucleolus V. TranslationVI. Genetic codeOutline of Current Lecture VII. ClickersVIII. tRNAa. Conformationsb. Necessary modifications IX. Wobble positionX. Aminoacyl-tRNAiXI. Process of Protein SynthesisXII. Eukaryotic vs. Prokaryotic RibosomesCurrent Lecture- Clicker Question 1: Why do we say that there are three reading frames?o The ribosome can “slip” over mRNAs and change the reading frameo There are three nucleotides in a codono One each to correspond to rRNA, tRNA and mRNA o All of the aboveo None of the above **Once ribosome is on the mRNA, it moves down it smoothly and it is very rare for it to “slip” anywhere on the mRNA - Clicker Question 2: Tryptophan has only one codon, UGG. So the anticodon found on tryptophan –tRNA is:o 5’-CCA-3’- mRNA is bound to the small subunit of the ribosome but is able to be read by the large subunit- tRNA: RNA adaptors that can recognize and bind to both codon and amino acid at different siteso Fold into precise 3-D structure  contains 4 short segments that are double helical, which fold into a clover leaf conformation (secondary conformation) Four loops are complementary with each other with non-complementary parts in betweenThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.o Undergoes further folding to create an L-shape structure, which is held together by additional H-bonds between different regions of the molecule (tertiary conformation)o The 2 regions at either end of the “L” are crucial One forms the anticodon: 3 nucleotides that complementary base pair with mRNA The other is a short ingle-stranded region at the 3’ end where amino acid matching the codon is attached- Since genetic code is redundant, some tRNA base pair with more than one codon and there is more than one tRNA for many of the amino acidso **Some are constructed so that only accurate base pairing is necessary in the first 2 positions, and a mismatch can occur in the third  known as the “wobble” positiono All of the four codons Ala, Gy, Pro and Val jave the same first two nucleotides and can also have any four of the nucleotides in the third position. “Wobble” position occurs when first two positions are selective and third can bind to whatever it pleases and still create the same amino acid- tRNAs have various modifications made to them. o tRNAs are synthesized by RNA polymerase III  usually start our larger and then are trimmed downo Some contain introns that must be spliced out (uses cut and paste mechanism instead offorming a lariat as in DNA splicing)o **Both require tRNA to be folded into 3-D conformation before leaving the nucleus - Recognition and attachment to correct amino acid depends on aminoacyl-tRNAi synthetase enzymes o Covalently couple each amino acid to its specific set of tRNA molecule, via hydrolysis of ATP  ***Hydrolysis is very exergonic, but the bond it creates is NOT HIGH ENERGY BONDo Correct amino has highest affinity for active-site pocket of its synthetase and is favored over the other 19o Second discrimination step: tRNA tries to force amino acids into a second pocket, which excludes correct amino acids and allows access to closely-related, incorrect ones. It hydrolyzes and release any amino acids that go into this pocket, increasing the accuracy of correct amino acids- Protein synthesis requires a peptide bond between the carboxyl group at the end of the growing polypeptide chain and free amino acid group on incoming amino acido Protein is synthesized step-wise from N-terminal to C-terminal end o Carboxyl end of growing chain remains activated by its covalent attachment to tRNA, which is broken by the addition of a new amino acido **Each amino acid carries with it the activation energy for the addition of the next amino acido **Free tRNA with charged amino acid must go to mRNA an be added to mRNA by ribosome. tRNA lined to C-terminal of mRNA - Protein synthesis is carried out in the ribosome: complex catalytic machine made from over 50 different proteins and several rRNAs to ensure accuracy and maintain the correct reading frame- Ribosomal subunits are assembled in nucleus, where the rRNAs associate with ribosomal proteins which have been transported into nucleus following their synthesis in the cytoplasmo Two ribosomal subunits are exported into the cytoplasm where they join together and synthesize proteins - Eukaryotic and prokaryotic ribosomes have similar designs: both have one large and small subunit that fit together; small unit allows tRNA to be paired to mRNA codons; large subunit catalyzes formation of peptide bonds to link together amino acids into polypeptide chaino They are also very different though: Prokaryotic ribosomes have smaller subunits, and smaller/fewer amounts of both proteins and RNA, making them less advanced then eukaryotic ribosomes- Two subunits only join together on an mRNA molecule, but are otherwise separate when inactive; ribosome releases mRNA when stop codon is reached and the two subunits separate again- Four binding sites: one for mRNA and 3 (E [ready to exit], P [attached to the polypeptide], and A ready for bond to form between C and N atoms]) for tRNAo tRNA only held tightly at A and P-sites if anticodon forms complementary base pairs withthe codon- Process of polypeptide chain formation:o tRNA with next amino avid binds to ribosome at A-site via base pairing with mRNA codono Carboxyl end of polypeptide chain is released from tRNA at P-site (by breaking bond between tRNA and its amino acid) and is joined onto free amino avid linked to tRNA at A-site, forming a new peptide bond; catalyzed by peptidyl transferaseo Large ribosomal subunit shifts so that two bound tRNAs are moved to E and P-siteso Conformational changes move small subunit 3 nucleotides in the same direction, so that ribosome is reset and ready to receive another amino acid and tRNA in E-site is released (EF1 and EF2 factors which hydrolyze GTP  GDP are responsible for the conformationalchanges)o Chain grows from amino to carboxyl