Name:
2 points
Chem 465
Biochemistry II
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)
B)
C)
D)
E)
1, 2, 3, 4
1, 2, 4, 3
2, 1, 4, 3
2, 3, 1, 4
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.
8. Eureka! I have discovered a novel bacterium that has a 21st 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
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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 6Aminoacyl-AMP
Class I
Step 2 aminoacylAMP 6Aminoacyl 2'-A on tRNA
Step 3 Aminoacyl 2'A on tRNA 6Aminoacyl-3'A on tRNA
Class II
Step 2 aminoacylAMP 6Aminoacyl 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
extended loop that are held together with a Zn2+ 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
hydrophillic on the other. Leu is every 7th 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.
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1 E 2B 3B 4C 5 C 6 A 7A
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