MCB124_ANSWERS_SET4 - MCB124 FALL 2008 Protein-DNA...

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MCB124 FALL 2008 Protein-DNA interactions ANSWERS TO PROBLEM SET4 page 1 I. PROTEIN_DNA INTERACTIONS A) 1) The consensus binding sequence is composed of the most probable bases in a selection experiment or in a sequence comparison. For this set, the preferred site size is a 14-mer, evidenced by the register within the 16-mer) G T T C x A t g T x G A A C (small letters designate "weak preferences" which may or may not be important). 2) Sorry, perhaps this is a overidealized example. The consensus sequence should represent the "best" binding sequence and the selected ones that are closer to it are therefore would be expected to bind the "best". In this case #3 and #8 are closest but 3, 6,8, and 10 are identical in the MAJOR bases. 3) The sequence is an inverted repeat. 4) Since sytA is an inverted repeat, it likely binds 2 repX monomer molecules. 5) # 4 is the deviant. It does nnot have any obvious similarity to the consensus. Deviant sequences happen for a variety of reasons i. Low specificity of the protein ii. Low stringency of the binding reaction iii. not enough selection-amplification cycles iv. deviants are selectively amplified by PCR v. too complex a library (too many random bases)- not seen here but in reality might be likely-given the large size of the site – the probability of an exact match for 10 major bases over at 14-mer window in a 16-mer is roughly 2 x 10 -8 = 4 -14* x 2 ** x 3 *** ( * probability of a unique 14-mer to occur within a 14 bp random sequence; ** factor of 2 for inverted repeat- two ways to get sequence; ** factor of 3 for 14-mer window ) This means that you need to screen at least ~10 8 different sequences, which is not too hard (a 35- mer weighs about 2x10 -19 g so 10 6 molecules weighs about 10 -13 g or 0.1 pg. However, the number of molecules will be much greater than that, affected by the following multipliers: N pcr = the minimal number of molecules needed to reliably PCR (10 3 -10 6 for regular people)- The 10 8 library will then weigh ~0.1 to 10 ug. E recovery -1 = the efficiency of recovery or what fraction of the number of representatives in the total library do you recover per binding cycle. E PCR -1 = the relative efficiency of PCR of the selected sequence vs the others. Bias = bias factor against your sequence in the original library due to different base coupling efficiencies during synthetic DNA synthesis. B) 6) In general, the backbone protection pattern can be said to identify the bases that are covered or contacted by the protein. In this case agGTTCAA (top strand) gtGTTCTA (bottom strand) 7) This is a thought question. Essentially, amino acids that make bidentate hydrogen bonds (Asn, Gln, Arg) are likely to be the "best" at making base- specific contacts but any H-bonding amino acid will do
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This note was uploaded on 04/06/2011 for the course MCB 124 taught by Professor Baldwin during the Summer '09 term at UC Davis.

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MCB124_ANSWERS_SET4 - MCB124 FALL 2008 Protein-DNA...

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