BCH5413 EXAM 3 WITH ALL QUESTIONS AND ANSWERS

What are the subunits of 70s and 80s ribosomes and their main functions? - ANSWER 70S has two subunits : 30S is the small submit that decodes mRNA and 50S is the subunit links amino acids together through peptide bonds 80S has two subunits: 40S and 50S: 40S small subunit, analogous to the 30S subunit in the prokaryotic ribosome 60S large subunit, analogous to the 50S subunit in the prokaryotic ribosome The ribosome consists of both ribosomal RNA and ribosomal proteins. Which part primarily determines the shape of the ribosome - ANSWER The structure of the ribosome is dependent on the ribosomal RNA and how they might base pair with each other at different segments. The ribosome consists of both ribosomal RNA and ribosomal proteins. Which part contains the peptidyl transferase activity? - ANSWER The 50S subunit (large subunit) contains the peptidyl transferase activity. The ribosome consists of both ribosomal RNA and ribosomal proteins. Which part contains the GTP hydrolysis activity? - ANSWER The 50S subunit (large subunit) contains GTPase activity. During protein synthesis, what kind of bond joins the amino acids? Which parts (functional groups) of the amino acids are bonded together? - ANSWER The 50S subunit links amino acids together through peptide bonds. Amino acids connect to form a peptide bond through a reaction between the amino group and carboxyl group. A peptide bond is formed through a dehydration reaction. This involves the removal of two hydrogens and an oxygen. tRNA Charging: How does the aminoacyl-tRNA synthetases recognize which tRNA gets its amino acid? - ANSWER Recognition will depend on nucleotides within the tRNA and binding sites within the protein. tRNA Charging: Describe the 2-step process for charging the tRNA. - ANSWER The first step is the activation of the amino acid with the help of ATP to form aminoacyl-AMP. The second step will use the energy from the aminoacyl-AMP bond to transfer the amino acid onto the acceptor stem of the tRNA. tRNA charging: Where does the amino acid get added to the tRNA? Be specific about the end of the RNA and the position of binding on the RNA. - ANSWER The high-energy bond between the aminoacyl and AMP will be used to transfer the amino acid to the 3' end of the tRNA; specifically, the amino acid will form an ester bond between the 3'carbon hydroxyl group on the adenosine of the tRNA and the carboxyl group of the amino acid. What is the wobble hypothesis? What is its significance? - ANSWER The wobble hypothesis is: The 3rd position base on a codon which is allowed to be different to accommodate different tRNA, this exception allows it to form a non Watson-Crick base pair with the anticodon. This is significant because the wobble allows some aminoacyl-tRNAs to pair with more than one codon True/False: Protein synthesis requires GTP. - ANSWER TRUE, When binding an aminoacyl-tRNA to the A site True/False: Wobble allows the different aminoacyl-tRNA to pair with same codon. - ANSWER TRUE, 3rd base of a codon is allowed to move slightly from it snormal position, allowing same aminoacryl-tRNAs to pair with more than one codon True/False: Ligase enzyme joins the amino acids to the respective tRNAs - ANSWER FALSE, AA attached by ester bond between 2'-OH of terminal adenosine of tRNA True/False: Aminoacylation of tRNAs requires GTP - ANSWER FALSE, 2 step process reuire AMP from ATP to activate AA, then,AMP is used to transfer the AA to tRNA True/False: Fmet-tRNA is the initiator tRNA in Eukaryotes - ANSWER FALSE, N-Formylmethionine=Fmet-tRNA is the initiator of Prokaryotes What differences are there between the ribosome binding in prokaryotes vs. eukaryotes? - ANSWER During translation in prokaryotes, the ribosome binds an operon at a ribosome binding site that precedes an open reading frame. Once the ribosome binds, there are various soluble factors that ensure the ribosome reads the correct code to make the correct protein.During translation in eukaryotes, the ribosome binds the 5' cap, then scans the mRNA for the AUG start site. The ribosome also requires soluble factors to initiate translation. What is the function of IF1? - ANSWER binds to the A site and keeps the fmet-tRNA from binding there What is the function of IF2? - ANSWER binds to the fmet-tRNA and GTP and brings the complex to the P site What is the function of IF3? - ANSWER binds to 30S subunit to keep it separate from the large subunit What is the function of hydrolysis of GTP after 70s initiation complex is formed? - ANSWER releases IF2 and GDP from the complex so polypeptide chain elongation can begin Explain the three steps in formation of 70s complex. - ANSWER The ribosome first dissociates. The 30S initiation complex is the first to form. IF3 binds to the 30s ribosome to prevent reassociation of the 2 ribosomal subunits. With the assistance of IF1 and IF2, IF3 binds to the Shine-Delgarno sequence (RBS) on the 5' end mRNA. This sequence is also recognized by a complementary sequence in the 16S rRNA.. IF2 will bind to the fmet-tRNA and to GTP and bring them to the P-site. No GTP hydrolysis occurs at this point. IF3 will leave the 30S initiation complex, allowing the 50S subunit to join the complex. At this point, GTP is hydrolyzed by the GTPase of the 50S subunit. This hydrolysis allows the release of IF2 (and GDP), leaving the fmet-tRNA in the P-site. At this point the 70S complex is complete and elongation can begin. Explain the three main steps of elongation. Be sure to indicate when GTP is needed and why. - ANSWER Once the 30S initiation complex is formed, IF3 comes off, allowing 50S subunit to bind. When 50S joins the 30S complex GTP is hydrolyzed to form 70S complex. GTP hydrolysis is carried out by GTPase in 50S subunit. The purpose of hydrolysis is to release IF2 and GDP from the complex , so the elongation step can begin How does translation stop? - ANSWER Translation stops when a stop codon is reached. These codons include UAA, UAG, and UGA. There are no tRNA that can recognize these codons so translation is terminated. What is indexing? - ANSWER Indexing is how the nucleotides are grouped within a frame, beginning at the P-site before translation. If the index is off by one nucleotide, the frame shifts, and the mRNA can be read differently, resulting in different amino acids being bound to the polypeptide chain and producing another protein. Premature stop codons can also result if indexing is not for the correct frame. Indexing: In general, describe how toe-printing allows researchers to determine the codon that is in the P-site? - ANSWER Toe-printing begins with artificial mRNA that contains the ribosome binding site. The mRNA would bind to the small ribosomal subunit, and a primer complementary to the 3' end of the mRNA is added. The primer would attach to the mRNA and elongate using reverse transcriptase until it reached the edge of the small ribosomal subunit. The primer length would change based on what nucleotides were bound to the P-site and were run on a sequencing gel. The longest band would mean that a Met was coded for, and AUG was in the P-site. If the indexing were off by one nucleotide, then a shorter band would appear, signaling that Cys was coded for. If the indexing were off by three nucleotides, then the shortest band would occur, which would mean a Phe was coded for. Toe-printing experiments showed that in order to ratchet to the correct reading frame, indexing requires ___________. - ANSWER Initiation factors, such as IF1, IF2, and IF3. How does mRNA secondary structure effect translation from a polycistronic message? - ANSWER by blocking translation. Ribosomes must bind to linear mRNA in order to proceed with translation, however, if an ORF is in the shape of say, a double-stranded helix, the ribosome will be unable to bind at this location. Therefore, there may be more proteins synthesized from one ORF that is linear and unblocked compared to another ORF on the same mRNA that is not in a linear shape. How do eukaryotic mRNAs become circular in the cytoplasm? Why is this important for translation? - ANSWER There are circular polysomes in eukaryotes. This is accomplished by the help from eIFs. eIF4E binds to cap and then binds eIF4G which then binds to PABPI which is bound to the poly A tail. This is important because more than 1 ribosome can translate a mRNA at one time. What is the advantage in regulating translation as well as