Study questions for required reading (newest first).
Study questions for Berns et al., 2004
- Why were plasmids expressing shRNA's used rather than transfecting in RNAi?
- The investiagtors validated the results of their initial screen in several ways.
How did they show that the siRNA's were (1) specific to their intended target genes and
(2) they actually act through p53-mediated pathways?
- What is the evidence from figures 2(c) and 3(a) that the five genes identified
mediated some but by no means all of p53's activities?
- The “bar-code” idea is interesting. In this analogy, what is the item
in which we are interested and what is the bar code that identifies it? What result
that was not observed would have made the bar code implementation here more
convincing? (Hint: how many shRNA's might one have expected to appear if the bar code
method had been applied as the initial screen?)
Study questions for Park et al., 2002
- How are RNAP complexes produced at a unique location on a template (figure 1A)?
These are called walking experiments. (Hint: What does “stalled by CTP
starvation” mean?)
- How was immobiization of the template on magnetic beads done? How did this allow
for efficient measurement of RNA release? What experiment confirms that the polymerase
as well as the RNA is released by Mfd (as was previously known)?
- What specific pattern in the gels of Figure 3 demonstrates that Mfd requires
upstream DNA to act on RNA polymerase?
- What accounts for the sensitivity of elongation complexes in Figure 2A to release?
Presumably they are “healthy” elongation complexes. What kind of kinetic
measurement could address this question?
- The model for Mfd-mediated rescue/release is that Mfd forces RNAP to move forward
and that the RNAP then either elongates or is dissociated. This should remind you of
our model for proofreading. What is the kinetic partitioning involved in the Mfd
system?
Study questions for Naktinis et al., 1996
- What is the basis of the “protein footprinting” assay used to
measure interactions among core, beta, and gamma?
- What is the evidence for competitive as opposed to simultaneous binding
of core and gamma to the beta sliding clamp?
- Figure 7 shows that the gamma complex can remove sliding clamps from DNA,
but the evidence is that the +gamma curve changes very little with time. Why
is much of the clamp still bound at the end of the reaction? What might the
gel filtration profile have looked like in the presence of a large excess of
linear DNA?
- What is the advantage to the cell in having primer-template DNA but not
nicked DNA stabilize the beta-core interaction? How does this play out during
the synthesis of an Okazaki fragment?
Study questions for Fischle et al., 2004:
- Define “epigenetic”, “marks”, “modules”,
“binary switch”, and “modification cassette” in the context
of this article. Note that the binary switch used here is quite different from the
computer science definition!
- The authors observe that acetyl-lysine sites are less likely than methyl-lysine
sites to be situated next to Ser or Thr, and this is an important element of
their “methyl/phos switch” hypothesis. What is their explanation
for the difference?
- What requirements are there for nucleosome stability if histone “marks”
are
to be epigenetic signals?
- If there are no histone demethylases, how might the methyl signal be turned
off permanently?
Study questions for Seeman et al., 1976:
- Why is it likely to be difficult for proteins to use all six of the major
groove recognition sites W1, W2, W3, W1’, W2’, and W3’ for
sequence-specific recognition?
- Why is the Table I entry for (G-C/C-G) discrimination at the S2’ position
a “(0)”? In other words, what is the basis for discrimination between
the two different base pairs, and why is it likely to be difficult?
- Inosine, which is the same as guanine except that the 2-NH2 group is replaced
by H, can be used as a probe for the groove recognized by a protein. Compare
the I-C base pair to the A-U and G-C base pairs and predict the result of an
experiment where G is substituted by I and the binding of either a major-groove
binding protein or a minor-groove binding protein is studied.
Study questions for Moser and Dervan, 1987:
- Why do we focus on these particular triple base pairs instead of any of
the legion of other possibilities? (Remember what’s important about
the particular Watson-Crick base pairs vs. all the others.)
- Why are the triplexes studied here stabilized by low pH? Why are they stabilized
by polyamines? Why is the increased stability of triplex at acidic pH not apparent
in Figure 5?
- Figure 3 demonstrates parallel orientation of the third strand oligo-T and
the homopurine strand in the duplex. What is the reasoning leading to this
conclusion? How does it rule out strand displacement as a mode of binding?
- If the third strand probe bound in the minor groove rather than the major
groove, what would the DNA-EDTA 9 footprinting histogram on the bottom right
of figure 4 look like, and why?
- Why do the shorter probes or mismatches in Fig. 4 cleave reasonably well
at low temperature but then cleave (and presumably bind) less and less effectively
as the temperature is increased?
- Why has it not been possible to generalize triple-strand recognition to
double-stranded targets of arbitrary sequence? In other words, why the restriction
to homopurine/homopyrimidine tracts?
Study questions for Watson and Crick, 1953:
- Does the WC structure most clearly resemble A, B, or Z form? Why?
- The authors state that "the two chains (but not the bases) are related
by a dyad perpendicular to the fibre axis." What is a "dyad," and
what does this statement suggest about whether the chains are parallel or antiparallel?
Under what circumstances would the bases in fact be related by a true dyad axis?
- Draw the preferred enol form of thymine (why is it preferred?). What would
enol-T base pair with? Why was it important to Watson and Crick that the keto
forms of the bases be preferred?
- Identify a nomenclature/numbering inconsistency between the WC paper and today's
labeling.
- The authors specifically suggest that their helical structure cannot apply
to RNA. Where/how do they say this?
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