Study Questions for Transcription/Translation/Protein Secretion
1) What does the core part of RNA polymerase do? What about the sigma subunit?
2) Why does sigma 70 recognize different promoters than the heat shock sigma factor does? What is the
purpose of having these different promoters? What does it mean that some promoters are poor matches to
consensus? What is the implication of this match for gene expression?
3) Unlike actively growing cultures of Bacillus subtilis old cultures (not growing) have lots of spores. It is
known that special sigma factors are expressed during sporulation. Consider a Bacillus strain that is always
expression gfp (SigA promoter) vs. a mutant where that promoter was changed to a SigF promoter. What would
the two Bacillus gfp strains look like under a fluorescence microscope during exponential phase of growth?
How about in late stationary phase? Note: the green fluorescence protein is very stable.
4) Draw a genetic map of a typical gene. Including the essential genetic elements enabling transcription and
translation to occur. Assume a Sigma 70 type promoter.
5) (i) At what sequences do ribosomes bind mRNA? (ii) Do you expect the following mRNA to be translated?
(iii) Where could translation begin on the mRNA? (iv) In general, can a ribosome bind at more than one site on
mRNAs? Note: AUG is the first codon.
5'- GGCCAGGAGGCUUCCAUGCGAUUGUUCAAGUGACA-3'
6) What would be the result (in terms of the produced protein) if RNA polymerase initiated transcription one
base upstream of its normal starting point and why? What would be the result (in terms of the produced protein)
if translation began one base downstream of its normal starting point and why?
7) In a polycistronic mRNA, would there be multiple promoters, or multiple ribosome binding sites or both?
Why?
8) What identifiable feature is present on secreted proteins compared to cytoplasm ones? What are the
components of this feature?
9) The N-terminal sequences of two secreted proteins are shown below. Which one will most likely be secreted
by TAT? Optional: use SignalP on the web to identify the n, h, and c regions. Where are the predicted cleavage
sites? http://www.cbs.dtu.dk/services/SignalP/ (make sure you select Gram negative bacterium)
DmsA: MKTKIPDAVLAAEVSRRGLVKTTAIGGLAMASSALTLPFSRIAHAVDSAIP
FhuD: MSGLPLISRRRLLTAMALSPLLWQMNTAHAAAIDPN
10) How can a pathogenic bacterium deliver a toxin (produced in its cytoplasm) directly into a eukaryotic cell?
11) How can proteins be translocated through the cytoplamic membrane? Do all proteins go through the same
secretion pathway through the cytoplasmic membrane?
12) How can a protein catalyze its own secretion from the periplasm and through the outer membrane? What
kind of protein secretion is this?
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