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FCH 532 Homework 7. . Put your name and student ID number on the top of each page. . Your
assignment will not be accepted if it is not legible.. . Indicate the phenotypes of the following E.
coli lac partial diploids in terms of inducibility and active enzymes synthesized.. I-P+O+Z+Y/I+P-O+Z+Y+. I-P+OCZ+Y-/I+P+O+Z-Y+. I-P+OCZ+Y+/I-P+O+Z+Y+. I+P-OCZ+Y+/IP+OC Z-Y-. . I-P+O+Z+Y-/I+P-O+Z+Y+. . This diploid has no promoter on the second segment
of DNA and no repressor or lac permease on the first. The phenotype in this case would be
inducible to produce the lac Z product in trans but would not be able to produce the lacY product
since this segment of DNA has no promoter. It could not respond to lactose since there is no way
to uptake this molecule into the cell.. . I-P+OCZ+Y-/I+P+O+Z-Y+. This diploid has no repressor
on the first segment of DNA with a constitutive operator. This segment will constitutively (non
regulated) produce the lacZ product. However, the production of the lacY product will be
regulated by the lac repressor. . . I-P+OCZ+Y+/I-P+O+Z+Y+. This diploid has no repressor on
either segment of DNA with a constitutive operator on the first with lacZ and lacY genes. This
phenotype will be constitutive expression of lacZ and lacY.. . I+P-OCZ+Y+/I-P+OC Z-Y-. There
are no functional genes associated with a promoter so there will be no production of either lacZ
or lacY in this mutant under any conditions.. . . . Superrepressed mutants, I`, encode lac
repressors that bind the operator but do not respond to the presence of inducer. Indicate the
phenotypes of the following genotypes in terms of inducibility and enzyme production. (a).
ISO+Z+ (b). ISOCZ+ (c). I+O+Z+/ ISO+Z+. . (a). ISO+Z+ . The phenotype here is that the lac
repressor cannot be removed from the operator so no induction of the lacZ gene.. . . (b). ISOCZ+
. This has an OC so the lacZ will be constitutively expressed.. . . (c). I+O+Z+/ ISO+Z+. Because
the lac repressor can be used in trans (that is, it is a diffusible factor), it does not have to be on
the same DNA sequence as the gene it is repressing. In this example, there will be no induction
of the lacZ gene since IS cannot be removed by the inducer from the operator.. . What is the
experimental advantage of using IPTG instead of 1,6 allolactose as an inducer of the lac operon..
. . IPTG will not degrade and has strong affinity for the lac repressor. This means that once it
binds, the lac repressor will be inactivated.. . . . Indicate the -10 region, -35 region, and the
initiating nucleotide on the sense strand of the E. coli tRNA Tyr promoter shown below.. . .
5’CAACGTAACACTTTACAGCGGCGCGTCATTTGATATGATGCGCCCCGCTTCCCGATA3’. 3’GTTGCATTGTGAAATGTCGCCGCGCAGTAAACTATACTACGCGGGGCGAAGGGCTA
T-5’. . What are the main differences between the prokaryotic RNAP and eukaryotic
RNAP?. Prokaryotic RNAP only has 5 subunits and the s factor. There is only one RNAP in
prokaryotes. Eukaryotes have 3 different RNAPs that have many more subunits than the
prokaryotic RNAP. The eukaryotic RNAPs recognize different promoters and have to be
recruited to those promoters by transcription factors. . . Short answers. Define the following
terms in two or three sentences. Be specific. . Inducible enzyme-enzymes that are synthesized at
rates that vary with the cell’s circumstances/environment. . Constitutive enzyme-enzymes
that are involved in basic cellular housekeeping functions and are synthesized at a constant rate..
Beta galactosidase-enzyme encoded by the lacZ gene responsible for the enzymatic cleavage of
lactose disaccharide to glucose and galactose. When lactose is present in high concentrations,
will form 1,6-allolactose, the inducer that binds to the lac repressor protein.. IPTGisopropylthiogalactoside-an analog of 1,6-allolactose that binds irreversibly to the lac repressor
preventing the protein from binding to the lac operator. . Basal level expression-low level
expression (uninduced expression).. Hfr-High frequency of recombination cells where the
transfer of the bacterial chromosome from an Hfr cell can be transferred to an F- cell.. F factorFertility factor. Plasmid that confers the ability to transfer genetic material from one bacterial
cell to another through F pili. . Bacterial conjugation-process through which bacterial cells
transfer genetic material to other cells via a cytoplasmic bridge between cells.. UP elementupstream promoter elements are specific nucleotide sequences that are found -40 to -60 bases
away from the promoter region of highly expressed genes. These are AT rich and bind to the
CTD of prokaryotic RNAP.. Pribnow box – Conserved sequence found in prokaryotic
promoters around -10 nt of the initiation nucleotide of the mRNA sequence. Usually has a
consensus sequence of : 5’-TAATAT-3’ . Shine Dalgaro region- In prokaryotes, a
purine-rich sequence of 3-10 nucleotides that is ~10 nucleotides upstream of the initiation codon.
This sequence is partially complementary to the pyrimidine-rich sequence at the 3’ end of
16S rRNA.. TATA Box– A conserved nucleotide sequence found in many eukaryotic
promoters of structural genes found -35 of the initiation nucleotide. Unlike the Pribnow box, it is
not necessary for RNA transcription but rather defines the initation codon for the mRNA..
Wobble Hypothesis (define it - not give an example)- hypothesis that describes how a tRNA can
recognize several degenerate codons. The first two codon-anticodon pairings are typical WatsonCrick base pairings and the third position codon-anticodon pairing, although similar in geometry
to a Watson-Crick pair, can have several conformations. This allows several non-Watson-Crick
base pairs such as U-G or I-A. If C or A occupies the third position of an anti-codon, they can
only base pair in a Watson-Crick geometry. If a U, G, or I occupies the third position, two or
three codons can be recognized (See Table 32-5).. Puromycin –an antibiotic that binds to the A
site of a ribosome and causes premature chain termination during translation. This antibiotic
looks like the 3’ end of the aminoacylated tRNA and will affect both prokaryotes and
eukaryotes. . Rifamycin-class of antibiotics produced by Streptomyces that inhibit prokaryotic
but not eukaryotic RNAPs.. a-amanitin-a bicyclic octapeptide toxin produced by the mushroom,
Amanita phalloides that binds to RNAP II and RNAP III in eukaryotes and dramatically slows
down RNA synthesis by binding to a funnel beneath the protein’s bridge helix. Likely
impedes the conformational change of this helix that is necessary for RNAP II and RNAP III
translocation. Does not affect RNAP I, prokaryotic, mitochondrial, or chloroplast RNAPs..
Amber Stop codon named after Bernstein (German for “Amber―) and sometimes called a
“nonsense mutation―. Basically encodes a stop codon instead of a functional codon for an
amino acid. Two other stop codon mutations are plays on the name (Amber) called Ochre and
Opal mutations. . Class II topoisomerase - enzyme responsible for supercoiling DNA and acts by
making double stranded breaks in the DNA, passing the helix through the gap and then resealing
the double helix. . Primase – enzyme responsible for making the RNA primers necessary for
DNA polymerase to initiate DNA synthesis.. Snurp– snRNP-small nuclear ribonucleoproteins
that facilitate the splicing of pre-mRNA in eukaryotes.. Sigma factor-subunit for the prokaryotic
RNA polymerase that is necessary for binding to the bacterial promoter sequence in the DNA to
initiate transcription.. Open complex-DNA template strand of transcription bubble is in a tunnel
formed by (((((’subunits lined with basic amino acids. . Closed complex-UP elements from
DNA are in contact with the CTD of RNAP.. Rho factor is a helicase that unwinds RNA-DNA
and RNA-RNA double helices dependent on the hydrolysis of NTPs. Requires a specific
recognition sequence (80 -100 nt that lack a stable secondary structure and have multiple C rich
regions, G poor regions) on the newly transcribed RNA upstream of the termination site.
Attaches to nascent RNA at recognition site and migrates in the 5’( 3’ direction until it
encounters RNAP paused at termination site and unwinds the RNA-DNA duplex that forms the
transcription bubble. This releases the RNA transcript.. Ribozymes, self catalytic RNA enzymes,
are often thought to use the lariat model to remove sections of RNA similar to that described for
intron removal in mRNA. Illustrate and describe the lariat model for intron removal.. What is
catabolite repression? Give an example. Catabolite repression-prevents the wasteful duplication
of energy-producing enzymes. Glucose is the carbon source of choice for E. coli, so if it is
present in large amounts, the bacterium will suppress the expression of genes encoding proteins
involved in other catabolites’ metabolism. This happens even when metabolites such as
lactose, arabinose, or galactose are present in high concentrations.. What is the PaMaJo
experiment? In the PaJaMo experiment-Hfr bacteria of genotype I+Z+ were mated to F- strain
with a genotype I-Z- in the absence of inducer while (-galactosidase activity was monitored. At
first, no (-galactosidase activity because the Hfr donors lacked inducer and F- recipients were
unable to to produce active enzyme. After 1 h conjugation when the I+Z+ enter the F- cells (galactosidase began and ceased after another hour. The donated Z+ gene entering the cytoplasm
of the I- cell causes constitutive expression of the (-galactosidase gene. After the I+ gene is
expressed (after 1 h) it represses (-galactosidase synthesis. Therefore, I must be a diffusible
product.Why was this experiment important for biochemistry? This experiment confirmed that
there were diffusible elements that could control transcription of genetic material in cells (how
the lac repressor works).. What are some structural similarities/motifs used in DNA binding?
Helix-turn-helix or HTH motifs associate with target base pairs mainly via side chains extending
from the second helix of the HTH motif (recognition helix). HTH motifs are observed in the lac
repressor, trp repressor, cI repressors, and Cro proteins from bacteriophages. Another type of
structural motif observed in DNA binding proteins are (-ribbons or two stranded anti-parallel bsheets. (-ribbons are found in the met repressor (MetJ).. . . . Ans key. . . . . . . . . . . .
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