Name:____________________
Chem 465
Biochem II
Test 3
Multiple choice 4 points each.
1.
W hich of the following are features of the wobble hypothesis?
A)
A tRNA can recognize only one codon.
B)
Som e tRNAs can recognize codons that specify two different am ino acids, although both
am ino acids are always nonpolar.
C)
The third base in a codon always form s a norm al W atson-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.
Form ation of the ribosom al initiation com plex for bacterial protein synthesis does not require:
A)
form ylm ethionyl tRNA fMet.
B)
initiation factor 2 (IF-2).
C)
GTP.
D)
m RNA.
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 m ixture of (1) ribosom es, (2) all the other tRNAs and am ino
acids, (3) all of the cofactors and enzym es needed to m ake protein in vitro, and (4) m RNA for hem oglobin,
where in the newly synthesized hem oglobin would the Ala from Ala-tRNA Cys be incorporated?
A)
wherever Ala norm ally occurs
B)
wherever Cys norm ally occurs
C)
wherever either Ala or Cys norm ally occurs
D)
wherever the dipeptide Ala-Cys norm ally occurs
E)
nowhere; this is the equivalent of a nonsense m utation
4.
Glycosylation of proteins inside the endoplasm ic 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-acetylglucosam ine.
5.
Indicate whether each of the following statem ents is true (T) or false (F).
_T_ Bacterial m RNA is broken down within a few m inutes of its form ation in E. coli.
_F_ Bacterial m RNA consists only of the bases which code for am ino acids.
_F_ Polysom es do not necessarily contain m RNA.
_F_ Bacterial m RNA norm ally occurs as a double-stranded structure, with one strand containing
codons, the other containing anticodons.
_T_ Bacterial m RNA can be translated while it is still being synthesized.
6.
"Housekeeping genes" in bacteria are com m only expressed constitutively, but not all of these
genes are expressed at the sam e level (the sam e num ber of m olecules per cell). W hat is the
prim ary m echanism responsible for variations in the level of constitutive enzym es?
A)
All constitutive enzym es are synthesized at exactly the sam e rate, but som e are
degraded faster than others.
B)
Different genes have slightly different prom oters, with different affinities for RNA
polym erase holoenzym e.
C)
The sam e num ber of m RNA copies are m ade from each gene, but these m RNAs are
translated at different rates.
D)
Som e constitutively expressed genes are m ore inducible than others.
E)
Som e constitutively expressed genes are m ore repressible than others.
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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 enzym es A-ase and B-ase,
respectively, and also includes P (prom oter) and O (operator) regions as shown.
W hen a certain com pound (X) is added to the growth m edium of E. coli, the separate enzym es A-ase and
B-ase are both synthesized at a 50-fold higher rate than in the absence of X. (X has a m olecular weight of
about 200.) W hich of the following statem ents is true of the operon decribed above?
A)
W hen RNA polym erase m akes m RNA 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 m RNA that will then be
translated into 4 different proteins.
D)
The 5' end of the m essenger from this operon will correspond to the right end of the
operon as drawn.
E)
The 3' end of the m RNA from the operon will correspond to the left end of the
operon as drawn.
8.
Transcription of the lactose operon in E. coli is stim ulated by:
A)
the presence of glucose in the growth m edium .
B)
binding of the repressor to the operator.
C)
a m utation in the repressor gene that decreases the affinity of the repressor for the
operator.
D)
a m utation in the repressor gene that increases the affinity of the repressor for the
operator.
E)
none of the above.
9.
W hich of the following statem ents is true of the attenuation m echanism used to regulate the
tryptophan biosynthetic operon in E. coli?
A)
One of the enzym es in the Trp biosynthetic pathway binds to the m RNA and blocks
translation when tryptophan levels are high.
B)
Attenuation is the only m echanism 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)
W hen 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 polym erase II.
B)
TATA boxes.
C)
other general transcription factors.
D)
coactivators.
E)
all of the above.
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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 m ust first attach an am ino acid to a t-RNA using
Am inoacyl-tRNA synthetase. Describe how this enzym e works. How does it recognize the proper t-RNA,
where does the am ino 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 com partm ents 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 m itochondira, 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 rem oved 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
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3. Describe as com pletely as you can how the ara operon is controlled in E coli.
Rough outline of operon
araC
AraO2
AraO1/Pc CRP araI PBAD AraB araA araD
2 promoters
PBAD transcribed left to right for structural genes
Pc transcibed right to left for ara C regulatory gene
Start with Pc
if not repressed makes a repressor protein
when repressor protein (araC) concentration >40/cell
this repressor protein binds at O1 to repress its own synthesis so remains
at 40/cell
When Glucose present and arabinose not present
1 repressor binds to each AraO2 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
PBAD
arabinase binds to repressor protein araC, it changes conformation
in new conformation binds as dimer at araI site and araO1 site
in this dimer conformation cannot make araI/araO2 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
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5. Describe the control of the Galactose m etabolism 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 UASG
Regulatory gene Gal4 binds to UASG as a dimer
in absence of Galatose Gal80P binds to Gal4 and prevents it from acting
as an activator
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
class
Bonus 5 points. W hat is the chem ical m akeup of the m agic spot, and where was it seen in Biochem istry
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.
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