Chem 465 Biochemistry II Name: 2 points Multiple choice (4 points apiece): 1. Which of the following is not true of trna molecules? A) The 3'-terminal sequence is -CCA. B) Their anticodons are complementary to the triplet codon in the mrna. C) They contain more than four different bases. D) They contain several short regions of double helix. E) With the right enzyme, any given trna molecule will accept any of the 20 amino acids. 2. In E. coli, aminoacyl-trna synthetases: A) activate amino acids in 12 steps. B) are amino acid-specific; there is at least one enzyme specific for each amino acid. C) fall into two classes, each of which attaches amino acids to different ends of the trna. D) have no proofreading activities. E) require a trna, an amino acid, and GTP as substrates. 3. Bacterial ribosomes: A) bind tightly to specific regions of DNA, forming polysomes. B) contain at least one catalytic RNA molecule (ribozyme). C) contain three species of RNA and five different proteins. D) have specific, different binding sites for each of the 20 trnas. E) require puromycin for normal function. 4. The pathway for polypeptides exported from E. coli includes the following steps, which occur in what order for correct export? 1. A chaperone, SecA, binds to the polypeptide. 2. A chaperone, SecB, binds to the polypeptide. 3. ATP is hydrolyzed by Sec A. 4. SecA pushes 20 amino acids of the polypeptide into the translocation complex. A) 1, 2, 3, 4 B) 1, 2, 4, 3 C) 2, 1, 4, 3 D) 2, 3, 1, 4 E) 3, 1, 4, 2
5. Small signal molecules that regulate transcription are not known to: A) cause activator proteins to bind DNA sites. B) cause repressor proteins to bind DNA sites. C) directly bind to DNA sites. D) prevent activator proteins from binding to DNA sites. E) release repressor proteins from DNA sites. 6. Which of the following statements about regulation of the lac operon is true? A) Glucose in the growth medium decreases the inducibility by lactose. B) Glucose in the growth medium does not affect the inducibility by lactose. C) Glucose in the growth medium increases the inducibility by lactose. D) Its expression is regulated mainly at the level of translation. E) The lac operon is fully induced whenever lactose is present. 7. Which one of the following statements about the transcription attenuation mechanism is true? A) In some operons (e.g., the his operon), attenuation may be the only regulatory mechanism. B) Sequences of the trp operon leader RNA resemble an operator. C) The leader peptide acts by a mechanism that is similar to that of a repressor protein. D) The leader peptide gene of the trp operon includes no Trp codons. E) The leader peptide is an enzyme that catalyzes transcription attenuation. Longer questions - 17.5 points each - You may skip any one. st 8. Eureka! I have discovered a novel bacterium that has a 21 amino acid called Zehfusine I am studying the t-rna that codes for this amino acid. I already know that the mrna sequences that code for this amino acid are UUA, UUU, and UUC. Now, based on what you have learned about t-rna structure, propose a model for the t-rna. In your model include such things as: overall structure (both secondary and tertiary), sequence at the 3' end, sequence at the anticodon, and any modified bases you might find in this structure. 70-90 residues Cloverleaf shape but extra arm is possible Sequence at 3' end - 5'CAA-3' Both the 3' end and the 5' end were probably longer in the original transcript and were cut down in processing Several characteristic base modifications in TøC arm (right side of cloverleaf), D arm (Left side of cloverleaf, and anticodon arm. If the DNA codon is UUA, UUU or UUC, then the anti codon on the trna is most likely (3')-AAI-(5') overall 3D shape is more L-like with anticodon at one end and Amino acid arm at the other -2-
9. Continuing with my discovery of the Zehfusine amino acid metabolism. The structure of Zehfusine is shown below : What other amino acids does it resemble? Now, based on what you know about how AminoAcyl-tRNA synthetases work, propose a mechanism for how the Zehfusine synthetase works to attach Zehfusine to its trna. In this mechanism be sure to include all the chemical steps included in the process, what distinguishes a class I from a class II Synthetase, what structural clues the synthetase would look for on the trna, and any proofreading steps that might be included to make sure the proper amino acid is placed on the correct trna. Resembles ILE or maybe VAL Since probably 1 trna, there is only 1 sythetase Synthetase binds on inside of L shape trna uses clues built on this surface rather than the anticodon to recognize the proper trna Mech Step 1 AA + ATP Aminoacyl-AMP Class I Step 2 aminoacylamp Aminoacyl 2'-A on trna Step 3 Aminoacyl 2'A on trna Aminoacyl-3'A on trna Class II Step 2 aminoacylamp Aminoacyl 3'-A on trna Since Zehfusine varies by just 1 or 2 methyl groups from VAL or ILE, there is probably proofreading in the above process. There is probably a separate site that binds and destroys both ILE-AMP and Val-AMP. There is also probably a site that recognizes the Zehfusine trna bound to ILE or Val and destroys those incorrect parings. 10. Tell me about the different signals that can be placed in a protein s sequence. What do they look like, what do they do, how does the cell recognize and utilize the sequence, can you see the sequence in the final protein? Signal to direct to rough ER ( lysosomal, membrane or secreted proteins) 13-36 AA, 10-15 hydrophobic Aas, 1 or 2 positively charged AA s near amino terminus, short polar sequence with small amina acids near the C-terminus where the signal will be cleaved from the rest of the protein. Signal sequence is recognized as is coming off the ribosome and is bound by SRP (signal recognition Particle) that stops synthesis until the SRP is bound to ER membrane by SRP receptor. Protein synthesis then resumes and protein in synthesized and inserted into the ER. As signal peptide reached the inside of the ER, the signal peptide is recognized and removed so it is not present in the completed protein Targeting to chloroplast or mitochondria is also through an N-terminal signal, but the nature of the signal is not discussed in the text. Here the protein is completely synthesized before a cytosolic chaperone binds the proteins and takes it to the appropriate organelle where it is transported into the organelle and the amino terminal signal removed. The signal to target a protein to the nucleus is located somewhere within the protein itself in a sequence call the NLS (nuclear Localization Seqeunce). It usually consists of 4-8 residues with several consecutive Arg or Lys resides. This sequence is recognized in the cytosol by the á -importin protein, which is then bound by the â- -3-
importin, and the complex as a whole is transported through a nuclear pore into the nucleus. Where the importins are released in a complicated recycling process. Bacteria also use signal sequences to target proteins for the inner or outer membranes or the periplasmic space. These signals are similar to those used in eukaryotic cells to target proteins to the ER or the mitochondira. Here is is thought that the signal sequences make the proteins fold slowly, so they are bound to the secb chaperones, which deliver the proteins to the seca protein on the inner surface of the bacterial membrane. The seca protein then uses ATP energy to puch the protein though the membrane in steps of about 20 AAs/ATP. 11. Tell me about the various domains found in DNA binding proteins. DNA binding domains Helix-turn-helix - used in most bacterial regulatory proteins. 7-9 residues in a helix, a â-turn and then another 7-9 residues of helix. Not stable by itself, so it must be a part of a larger structure Zinc Finger - Used in most Eukaryotic DNA binding proteins - 30 AA s in a long 2+ extended loop that are held together with a Zn ion complexed with 4 Cys or 2Cys and 2 His. Homeodomain - used in Eukaryotic cells particularly for development genes. 60 residues that resembles the helix-turn-helix motif, but larger. Protein-Protein binding domains Leucine zipper - Extended helix with hydrophobic residue on one side and th hydrophillic on the other. Leu is every 7 residue. Hydrophobic side of one helix on one protein will interact with hydrophobic side of a helix on another protein. Basic Helix-loop-helix --50 residue sequence that forms two short amphipathic helices separated by a short loop. Used to bind to a similar sequence on a second protein Also mentioned in class Glutamine rich, Proline rich, and acidic domains 12 The synthesis of Zehfusine requires 5 different enzyme activities. Assuming that the Zehfusine metabolism is controlled much like other amino acids in bacteria, propose a structure for the Zehfusine operon and describe how this operon works. mrna for all five genes one after another on the DNA with a single promoter upstream. There is also a separate gene for a repressor gene that codes for a Zefusine repressor protein. This protein binds to the Zehfusine promoter and represses the expression of the gene unless Zehfusine is readily available to bind to the repressor to release it from the promoter. The early part of the zefusine also contains an attenuator sequence that codes for a short peptide that contains 4 zefusine residues and some hairpin loops followd by a polyu sequenfe. When zefusine is present the ribosome moves quickly through this region and hairpin forms that induces the RNA polymerase to fall off the DNA in a rho-independent manner. When [zehfusine] is low the ribosome stalls, and a different set of hairpins form, so the RNApolymerase remains on the DNA and transcribes the entire gene. -4-
1 E 2B 3B 4C 5 C 6 A 7A -5-