Homework #3 KEY - answer key PDF

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Agro/ANSC/Gene/Hort/Bio 305 Fall, 2018 HW#3 (Due back on Nov 2) 1. In which of the ribosomal sites, the A site, P site, and/or E site, could the following be found? A. A tRNA without an amino acid: E site, (P site, transient)

B. A tRNA with a polypeptide attached: P site (A site, transient)

C. A tRNA with a single amino acid attached: A site

D. A tRNA with fMet: P site Q2. The following DNA sequence occurs in the nontemplate strand of a structural gene in bacterium (the promoter sequence is located to the left but is not shown): 5’-GAATGTCAGAACTGCCATGCTTCATATGAATAGACCTCTAG-3’ a) What is the ribonucleotide sequence of mRNA molecule that is transcribed from this piece of DNA? 5’-GAAUGTCAGAACUGCCAUGCUUCAUAUGAAUAGACCUCUAG-3 b) What is the amino acid sequence encoded by this mRNA? Indicate both the amino terminal and carboxy terminal ends. +NH

2-

Met-Ser-Glu-Leu-Pro-Cys-Phe-Ile-COO-

3. Transcriptional regulation often involves regulatory proteins that bind to a segment of DNA and small effector molecule that binds to a regulatory protein. Do the following terms apply to a regulatory protein, a segment of DNA or a small effector molecule? a. Repressor

RP

b. Inducer

E

c. Operator

D

d. Corepressor

E

e. Activator

RP

f. Attenuator

D

g. Inhibitor

E

h. CAP site

D

i. lacZ

D

j. cAMP

E

k. Allolactose

E

l. Tryptophan

E

m. Enhancer

D

n. Mediator

RP

Q4. Drawn below is an outline of the Lac operon. Label the lac operon and describe the function of each element on the operon. The arrows point to the different elements/genes in the operon.

_______________________________________________________________________ _______________________________________________________________________

1

2

3

4

5

6

7

1.

CAP site: Site for binding of activator

2.

lacP: Promoter for lac operon

3.

lacO: Site for Repressor binding

4.

lacZ: -galactosidase Enzyme catalyzing the breakdown of lactose to glucose and galactose; Conversion of lactose into allolactose

5.

lacY:

6.

lacA: Galactosidase acetylase Modifies lactose by acetylation

7

lac terminator

Lactose permease Uptake of lactose

Q5. How is the lac operon regulated when E. coli is grown both in the presence of glucose and lactose? -

CAP is an activator protein of the lac operon. It binds to the activator site when cAMP levels are high. Glucose inhibits cAMP levels, thereby inhibiting transcription of the lac operon.

Q6. What is the status of the trp operon in the following figure? Give an explanation for your answer?

The ribosome at the stop codons stalls, with the result that regions 1 and 2 are blocked and are not able H-bond with each and neither can region 2 H-bond with region 3. Thus regions 3 and 4 H-bond and form the terminator. The RNA polymerase is released and the transcription of the whole operon is terminated. This is the status of the operon under conditions of high Trp in the cells.

Q7. Transcriptional control of eukaryotic gene expression can be exquisitely specific: It will be specifically transcribed in some tissues at very defined times. Explore how this specificity arises by addressing the following questions: a. Distinguish between the functions of core promoter and enhancers in transcriptional regulation. The core promoter is the site where RNA polymerase, with the help of basal transcription factors, binds and initiates transcription about 25 bp downstream.

b. Distinguish between the functions of general or basal transcription factors and specialized or regulatory transcriptional factors in transcriptional regulation. General transcription factors recruit RNA polymerase to the TATA box, which in turn initiates transcription Regulatory transcription factors – activators /repressors, bind to the regulatory sites, enhancers/silencers and influences the rate of transcription factors

c. Give an example of how a regulatory transcription factor is activated. Transcription factor can be regulated as follows: ▪ Association with an effector molecule ▪ Protein – protein interaction ▪ Covalent modification Q8. Gene expression in eukaryotes is regulated at steps beyond transcription. Briefly, describe these steps and give the mechanism of regulation. Posttranscriptional regulation: i. Alternative splicing involving splicing factors that regulate which exons are involved in determining which exons are included in the resulting mRNA. The splicing factors may be regulated by binding to effector molecules, covalent modificator or protein-protein interaction ii.

RNA stability mediated by sequences in the 3’UTR like the AU-rich region involving some kind of RNA binding proteins.

iii.

Antisense RNA

iv.

RNA interference involving microRNA (miRNA) or short-interfering RNAs (siRNA). Inhibit translation of RNA or causes mRNA degradation

v.

Phosphorylation of ribosomal initiation factors.

vi.

Secondary structures in the 5’UTR along with specific RNA binding proteins that can regulate translation...