Prokaryotic Physiology. March 3, 2017

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Jodie Cook
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1 1. (10 pts) Explain the replication of both strands of DNA in prokaryotes. At a minimum explain the direction of synthesis, synthesis of the leading and lagging strand, separation of the strands and the fidelity of replication. You do not need to explain the initiation of replication. That is a separate question below. Synthesis 5 ->3 Helicase unwinds strands Topoismerase I or II unwinds supercoiling in front of Helicase DNA Pol III holoenzyme (two DNA Pol III) DNA Pol has 3 ->5 proof-reading activity Okazaki fragments DNA Pol I ligase Primase RNA primer RNase H

2 2. (10 pts) Explain how DNA replication is initiated in prokaryotes. Ori C site 30 DnaA binding 45 bp AT-rich region SSB DnaB (helicase) HU protein 3. (5 pts) E. coli growing with a generation time of 80 mins. has a G 1, C and D phase of 20 min, 40 min, and 20 min, respectively. Define G 1, C and D. How is it possible for E. coli to grow with a generation time as short as 20 min? G1- gap phase; cells carry on basic metabolism and maintenance. variable in length. C DNA synthesis phase; invariant D Division phase; cells grow and divide at the end of this phase; invariant Cells eliminate the G1 phase and overlap the C and D phase. This results in multiple copies of the genome in a single cell.

4. (10 pts) Below are the results of an experiment by Meselson and Stahl. Explain why the experiment was done, how the experiment was conducted and the significance of these results. 5.

3 4. (10 pts) Below are the results of an experiment by Meselson and Stahl. Explain why the experiment was done, how the experiment was conducted and the significance of these results. 5. Meselson and Stahl were evaluating the hypotheses that DNA replication was conservative, semi-conservative or dispersive. They grew E. coli for many generations in a medium containing heavy nitrogen (N 15 ) which the cells used to produce heavy DNA (the band at generation 0). They then added the cells to a medium containing only light nitrogen (N 14 ). After one generation all of the bacteria contained DNA that was composed of one half heavy nitrogen and one half light nitrogen. After two generations the DNA consisted of doublestranded DNA that was composed entirely of light nitrogen or double-stranded DNA that was one half light nitrogen and one half heavy nitrogen. In subsequent generations the amount of entire light nitrogen DNA increased. Thus, they demonstrated the DNA replication is semiconservative (they falsified the conservative and dispersive hypotheses).

6. (5 pts) Below is a bacterial growth curve. Label the growth phases on the graph. What is the generation time for this bacterium under these conditions?

4 6. (5 pts) Below is a bacterial growth curve. Label the growth phases on the graph. What is the generation time for this bacterium under these conditions? How many generations have passed during the time it takes for the population to go from 2X108 CFUs/ml to 8x109 CFUs/ml (round to the nearest tenth)? Label lag, log and stationary phases. To determine generation time simply choose points on the log portion of the y-axis, one of which is twice the other, and determine from the graph roughly how many minutes have passed. For example, how long did it take to go from 1x10^8 CFUs/ml to xx10^8 CFUs/ml? It took from roughly 2.75 h to 3.25 h or ( = 0.5 h) 30 mins. Number of generations = (log 8x10^9-log 2X10^8)/log 2 = ( )/0.301= 1.6/0.301=5.3

5 7. (10 pts) Describe or diagram the components of RNA Polymerase. How are different promoter sequences recognized? How does a RNA Polymerase recognize when to stop transcribing a gene? Two alpha subunits, a beta subunit, a beta prime subunit and a sigma factor. The sigma factor provides specificity for different promoter sites. Most genes utilize a sigma 70 promoter site which has a TAATA (-10) and TTGACA (-35) consensus sequence. Transcription stops in either a Rho-dependent process or Rho-independent process. Rhodependent termination requires a Rho protein to recognize a Rut (Rho-utilization) site of ~70 nt, C-rich region downstream of the transcribed region. The Rho protein displaces the RNA Pol in an ATP dependent reaction. Rho-independent termination is the result of the formation of 7-20 bp GC rich stem-loop structure in the RNA that stalls the RNA Pol. Eventually the RNA Pol dissociates from the DNA.