Ans key
FCH 532 Homework 7
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1.
Indicate the phenotypes of the following E. coli lac partial diploids in terms of inducibility and active enzymes synthesized. a.
I
-
P
+
O
+
Z
+
Y
-
/I
+
P
-
O
+
Z
+
Y
+ b.
I
-
P
+
O
C
Z
+
Y
-
/I
+
P
+
O
+ c.
I
-
P
+
O
C
Z
+
Y
+
/I
-
P
+
O
+ d.
I
+
P
-
O
C
Z
+
Y
+
/I
-
P
+
O
C
Z
-
Y
+
Z
+
Y
+
Z
-
Y
a.
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. b.
I P + O C Z + 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. c.
I P + O C Z + 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. d.
I + P O C Z + Y + /I P + O C 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.
2.
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).
I S O + Z + (b).
I S O C Z +
(c).
I
+
O
+
Z
+
/ I
S
O
+
Z
+
(a). I S O + Z +
The phenotype here is that the lac repressor cannot be removed from the operator so no induction of the lacZ gene.

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(b). I S O C Z +
This has an O C so the lacZ will be constitutively expressed.
(c). I + O + Z + / I S O + 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 I S cannot be removed by the inducer from the operator.
3.
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.
4.
Indicate the -10 region, -35 region, and the initiating nucleotide on the sense strand of the E. coli tRNA Tyr promoter shown below.
5’-CAACGTAACACTTTACAGCGGCGCGTCATTTGATATGATGCGCCCCGCTTCCCGATA-3’
3’-GTTGCATTGTGAAATGTCGCCGCGCAGTAAACTATACTACGCGGGGCGAAGGGCTAT-5’
5.
What are the main differences between the prokaryotic RNAP and eukaryotic
RNAP?
Prokaryotic RNAP only has 5 subunits and the

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.
A.
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.

IPTG-isopropylthiogalactoside-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).

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
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 factor-Fertility 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 element-upstream 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 Watson-Crick base pairings and the third position codonanticodon 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.
 
-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.

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
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 + enter the F- cells

-galactosidase began and ceased after another hour. The
Z + 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

Ans key 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 b-sheets.

-ribbons are found in the met repressor (MetJ).