Chem 465 Biochem II Test 3 Name: Multiple choice 4 points each. 1. Which of the following are features of the wobble hypothesis? A) A trna can recognize only one codon. B) Some trnas can recognize codons that specify two different amino acids, although both amino acids are always nonpolar. C) The third base in a codon always forms a normal Watson-Crick base pair. D) The "wobble" occurs only in the first base of the anticodon. E) A naturally occurring trna exists in yeast that can read both arginine and lysine codons. 2. Formation of the ribosomal initiation complex for bacterial protein synthesis does not require: A) formylmethionyl trna fmet. B) initiation factor 2 (IF-2). C) GTP. D) mrna. E) EF-Tu. 3. It is possible to convert the Cys that is a part of Cys-tRNA Cys to Ala by a catalytic reduction. If the resulting Ala-tRNA Cys were added to a mixture of (1) ribosomes, (2) all the other trnas and amino acids, (3) all of the cofactors and enzymes needed to make protein in vitro, and (4) mrna for hemoglobin, where in the newly synthesized hemoglobin would the Ala from Ala-tRNA Cys be incorporated? A) wherever Ala normally occurs B) wherever Cys normally occurs C) wherever either Ala or Cys normally occurs D) wherever the dipeptide Ala-Cys normally occurs E) nowhere; this is the equivalent of a nonsense mutation 4. Glycosylation of proteins inside the endoplasmic reticulum does not involve: A) dolichol phosphate. B) an Asn residue on the protein. C) a His residue on the protein. D) glucose. E) N-acetylglucosamine. 5. Indicate whether each of the following statements is true (T) or false (F). _T_ Bacterial mrna is broken down within a few minutes of its formation in E. coli. _F_ Bacterial mrna consists only of the bases which code for amino acids. _F_ Polysomes do not necessarily contain mrna. _F_ Bacterial mrna normally occurs as a double-stranded structure, with one strand containing codons, the other containing anticodons. _T_ Bacterial mrna can be translated while it is still being synthesized. 6. "Housekeeping genes" in bacteria are commonly expressed constitutively, but not all of these genes are expressed at the same level (the same number of molecules per cell). What is the primary mechanism responsible for variations in the level of constitutive enzymes? A) All constitutive enzymes are synthesized at exactly the same rate, but some are degraded faster than others. B) Different genes have slightly different promoters, with different affinities for RNA polymerase holoenzyme. C) The same number of mrna copies are made from each gene, but these mrnas are translated at different rates. D) Some constitutively expressed genes are more inducible than others. E) Some constitutively expressed genes are more repressible than others. 1
7. The diagram below represents a hypothetical operon in the bacterium E. coli. The operon consists of two structural genes (A and B), which code for the enzymes A-ase and B-ase, respectively, and also includes P (promoter) and O (operator) regions as shown. When a certain compound (X) is added to the growth medium of E. coli, the separate enzymes A-ase and B-ase are both synthesized at a 50-fold higher rate than in the absence of X. (X has a molecular weight of about 200.) Which of the following statements is true of the operon decribed above? A) W hen RNA polymerase makes mrna from this operon, it begins RNA synthesis just to the left of gene A. B) The repressor for this operon binds just to the right of A. C) All four genes (A, B, O, and P) will be transcribed into an mrna that will then be translated into 4 different proteins. D) The 5' end of the messenger from this operon will correspond to the right end of the operon as drawn. E) The 3' end of the mrna from the operon will correspond to the left end of the operon as drawn. 8. Transcription of the lactose operon in E. coli is stimulated by: A) the presence of glucose in the growth medium. B) binding of the repressor to the operator. C) a mutation in the repressor gene that decreases the affinity of the repressor for the operator. D) a mutation in the repressor gene that increases the affinity of the repressor for the operator. E) none of the above. 9. Which of the following statements is true of the attenuation mechanism used to regulate the tryptophan biosynthetic operon in E. coli? A) One of the enzymes in the Trp biosynthetic pathway binds to the mrna and blocks translation when tryptophan levels are high. B) Attenuation is the only mechanism used to regulate the trp operon. C) Trp codons in a leader peptide gene allow the system to be sensitive to tryptophan levels in the cell. D) When tryptophan levels are low the trp operon transcripts are attenuated (halted) before the operon's structural genes are transcribed. 10. Eukaryotic basal (general) transcription factors can bind specifically to: A) RNA polymerase II. B) TATA boxes. C) other general transcription factors. D) coactivators. E) all of the above. 2
Essay questions. 15 points each. Do any 4- If you do all 5 I ll give you the best 4 1. Before you can begin to synthesize a protein, you must first attach an amino acid to a t-rna using Aminoacyl-tRNA synthetase. Describe how this enzyme works. How does it recognize the proper t-rna, where does the amino acid get attached to the t-rna, what is the energy cost of this process, is there any proofreading built into this process? Usually 1 synthetase for each AA, even if >1 codon Recognizes the correct t RNA,not by antidocon, but by structure of amino acid arm itself. Even then it doesn t need the whole arm, but a single base pair at the proper place in the arm Overall rxn scheme ATP and AA bind in active site, and AA linked by COO end to AMP PPi released depending on mech Class I mech- AA transfered to 2' O of CCA terminal ribose Then shifted over to 3' O Class II mech Transfered directly to 3'O While only used 1 ATP for synthesis, the release of PPi is equivalent to use of 2 ATP s of E Yes there is proofreading in a second independent site that is designed to hydrolyze incorrect AA-tRNA s Actually proven in only a few sythetases, but not necessarily all Will bind incorrect AA-AMP and hydrollyze, so doesn t get to second step 2. During protein synthesis, proteins are targeted toward certain compartments using signal sequences. Describe what the signal sequences are for a protein that is to be used in the cytosol, one that is to be sent to the mitochondira, one that is to be exported from the cell, and one that is to go to the nucleus. In which of these cases are the signal sequences removed during protein processing. Cytosol no sequence simply made in cytosol and dumped there NLS nuclear localization sequence, located within the protein sequence not given in text Signal not removed so protein may be relocated after nucleus envelop broken down during cell division Mitocondria 20-35 AA at N terminal end rich in Ser Thr and basic removed as transported into inner matrix of Mitochondria Rough ER and tranport out of cell 13-36 aa at n-terminal end usuallly 1 or 2 + charge at N terminus followed by 10-15 hydrophobic Then a few polar and cleavage site at Ala or Gly removed as tranported across ER membrane 3
3. Describe as completely as you can how the ara operon is controlled in E coli. Rough outline of operon arac AraO AraO /P CRP arai P AraB araa arad 2 1 c BAD 2 promoters P BAD transcribed left to right for structural genes P transcibed right to left for ara C regulatory gene c Start with P c if not repressed makes a repressor protein when repressor protein (arac) concentration >40/cell this repressor protein binds at O 1 to repress its own synthesis so remains at 40/cell When Glucose present and arabinose not present 1 repressor binds to each AraO 2 and arai then protein-protein interaction between these 2 repressors make loop in DNA represses transription of arabad When Glucose absent and arabinose present Glu low, camp high, camp bind to CRP and CRP bind to binding site to left of P BAD arabinase binds to repressor protein arac, it changes conformation in new conformation binds as dimer at arai site and arao 1 site in this dimer conformation cannot make arai/arao 2 loop, so polymerase has free access to promoter site, as well as stimulation by presence of CRP 4. Describe how attenuation control works in the trp operon of E.coli. Diagram like figure 28-23 from text would help relies on close coupling between RNA synthesis by RNA polymerase and protein synthesis by ribosome mrna for trp operon starts with sequence of a short peptide only about a dozen AA, but 2 in a row are Trp has standard stop Has hairpin to signal for RNA polymerase to terminate synthesis Also has an alternative hairpin structure that messes up the hairpin used to stop RNA syntheis. so when [Trp] high, reads and synthesizes peptide, and ribosome covers the alternate haripin before it can form to mess up the normal termination hairpin. Thus proper hairpin forms and RNA polymerase drops off DNA before before RNA message can extend into the structural genes for tryptophan synthesis. When [Trp] low ribosome gets to peptide with 2 trp in a row and stalls because can t get charged trna to make peptide. As RNA polymerase pulls ahead the alternate hairpin forms, preventing the formation of the termination hairpin. Without this signal the RNA polymerase stays on the DNA and continues to elongate the mrna to include the structural genes of the trp operon 4
5. Describe the control of the Galactose metabolism genes in yeast. Several structural gene spread out on different chromosomes 3 major regulatory genes Gal 3, Gal4, and GAL80, also on different chromosomes All structural genes have a normal TATA box and Inr region, and upstream activator called UAS G Regulatory gene Gal4 binds to UAS G as a dimer in absence of Galatose Gal80P binds to Gal4 and prevents it from acting as an activator class In presence of Galatose Galactose binds to Gal3P Gal3p binds to GAL80P Binding of GAL3p to GAL80p allows Gal4 to act as activator Now HMG protein allow DNA to bend SWI/SWF and/or SAGA start histone remodeling Mediator and/or TFIIA may help binding of RNApolI to TATA box Also is control to turn entire system off if Glucose is present, but didn t go into in Bonus 5 points. What is the chemical makeup of the magic spot, and where was it seen in Biochemistry Magic spot is ppgpp or ppgppp Produced by bacterial ribosomes when they can t get enough charged t-rna s for protein synthesis. Used as a signal to shut down synthesis of RNA. 5