RNA Polymerase / Promoter Interaction E. in coli HOLOENZYME CORE ENZYME " ## RNA bp 5-6 bp. template strand [-35] [-10]

Transcription

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2 RNA Polymerase / Promoter Interaction E. in coli HOLOENZYME CORE ENZYME " ##! "' 5' 3' TT GACAT AACTGTA RECOGNITION bp 5-6 bp TATAAT AT AT TA UNWINDING [-35] [-10] 5' template strand RNA

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4 Note bacteriophage promoters. Strong vs Weak Promoters

5 The Lac Promoter (P LAC ) is Weak Comparison of the E. coli!70 consensus promoter seq uence (top) with the Lac Promo ter (bottom ) bp - T T G AC ANN NN NN NN NN NNN NN NN T A T A AT T T T AC AN NNN NN NN NNN NN NT AT G T T - 14 bp -

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7 CODON BIA S IS CORR ELATED WI TH trna ABUNDANCE Rel ative Abundance of Yeast Leucine Co dons in Highly Expressed Genes 5' UUA UUG CU U CU C CU A CU G 3' 3' AA U AAC GAG GA U 5' Rel ative abundance of Yeast Leucine trna's

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9 in E. coli Charging: Met + ini@ator trna ini@ator trna Met

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12 A G U C 3' CODON U C A G I

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15 A repair enzyme, Uracil DNA glycosylase, can excise U from the GU mispair by cleaving the glycoside bond. The resulting AP site is then a substrate for the AP endonuclease excision and ss gap reapair pathways (discussed in the text) which restore the original target GC. The text also briefly notes the possibility that T ocurrs in DNA, rather than U, because it creates a context for efficient repair of AU lesions. (See discussion of 5-meC below.)

16 Excision Repair of GU mismatch.

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19 NITROUS ACID AT - > GC GC - > AT NOT MUTAGENIC

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23 NO!

24 What effect?

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26 It depends on the of the lesion, and on which repair mechanism you are talking about.

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28 please go over the reca gene and how it is used in post replica6ve repair and the process you were discussing in lecture on tuesday? Thank you! RecA DNA Polymerase V

29 Could you please review the SOS response Stalled fork (at dimer in template strand) of SOS Response Polymerase V inserts random bases opposite dimer leading to transi@ons + transversions.

30 what is the significance of having IS elements oriented in the same or opposite direc6on? None that I am aware of.

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34 TRANSDUCTION

35 Generalized Transduction Note the similarities to transformation.

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37 Phage Lambda and Specialized Transduction Specialized transduction requires a "temperate" or "lysogenic" bacteriophage whose genome can be inserted into the host cell genome by recombination. Ordinarily, the recombination is at a defined location. Specialized transduction also subsequently requires the rare incorrect excision of the viral genome. Therefore, the phage genome carries a small segment of the host genome originating from the boundary of the phage integration site. This situation is much different than that of Generalized Transduction. In this section do not worry about they details of Lambda dgal and helper phages.

38 INTEGRATION

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43 3.Which one of the following shows a sequence that is consistent with the typical sequences found at the ends of IS elements?

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50 CH 3 5' p C p C p G p G p 3'! 3' p G p G p C p C p 5' 5' p C p C p G p G p 3'! 3' p G p G p C p C p 5' CH 3 5' p C p C p G p G p 3'! 3' p G p G p C p C p 5' CH 3 CH 3 CpG Methyltransferase

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52 Co- Dominance by PROBE

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54 riflop or riflip

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58 3.A certain gene from E. coli, gene X, codes for a protein that has SERINE (Ser) at amino acid posi@on 100. The en@re protein is 200 amino acids long. E. coli DNA is digested with the restric@on endonuclease BglII (5' AGATCT 3') and the fragments are separated on an agarose gel. The BglII DNA fragments are transferred to a nylon membrane and then probed with a radioac6ve probe complementary the en6re sequence of gene X. This Southern Analysis reveals two fragments of 1.6 and 0.9 kb.in a different strain of E. coli, a single base pair subs6tu6on has caused a missense muta6on such that SERINE is replaced by PROLINE (Pro) at posi6on 100 in the protein. If DNA from this strain is subject to Southern analysis by the same procedure as above, a single band of 2.5 kb is found.a. What amino acid is at posi6on 99 of the protein in the original strain? B. What amino acid is at posi6on 99 of the protein in the mutant strain? C. Is it possible to iden6fy which Serine codon is used in the original strain, and if so which is it? D. Determine the iden6ty of the base pair change responsible for this RFLP. E. Is it possible to iden6fy which Pro codon is used in the new mutant strain, and if so which is it?a. Arg B. Arg C.5' UCU 3' D.5' CCU 3' E.T A- - - >CG(=A T- - - >GC)a6irstposi<on

59 A Minimum Set of Ala trna s

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62 19.1 How can inducible and repressible enzymes of microorganisms be ANS: By studying the synthesis or lack of synthesis of the enzyme in cells grown on chemically defined media. If the enzyme is synthesized only in the presence of a certain metabolite or a par@cular set of metabolites, it is probably inducible. If it is synthesized in the absence but not in the presence of a par@cular metabolite or group of metabolites, it is probably repressible.

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64 6.20 Feedback Inhibition of Metabolic Pathways Non-

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68 Yes