Deck 28: Regulation of Gene Expression

Question 1

The active form of lactose repressor binds to the operator with a dissociation constant of 10 13 M for the reaction R + O RO. About 10 molecules per E. coli cell suffice to keep the operon turned off in the absence of inducer.
(a) If the average E. coli cell has an intracellular volume of 0.3 × 10 12 mL, calculate the approximate intracellular concentration of repressor.
(b) If the average cell contains two copies of the lac operon, calculate the approximate intracellular concentration of operators.
(c) Calculate the average intracellular concentration of free operators under these conditions.
(d) Explain how a cell with a haploid chromosome could contain an average of two copies of the lac operon.

Answer 1

(a)The approximate concentration of intracellular repressor would be as follows: (b)The approximate concentration of intracellular operators would be as follows: (c)The approximate intracellular concentration of the free operators would be approximately .
(d)A rapidly growing cell possess two copies as the cell during division divides the two sister chromatids of the chromosome. These sister chromatids replicate and form two complete set of chromosomes again.

Question 2

Is attenuation likely to be involved in eukaryotic gene regulation
Briefly explain your answer.

Answer 2

The chances of attenuation (weakening) by direct coupling of transcription and translation is absent in eukaryotic cells. This is because the process of transcription and translation occurs in different compartments of the cell hence the chances of direct coupling are zero. The transcription occurs in the nucleus and the translation occurs in the cytosol.

Question 3

Suppose you want to study the transcription in vitro of one particular gene in a DNA molecule that contains several genes and promoters. Without adding specific regulatory proteins, how might you stimulate transcription from the gene of interest relative to the transcription of the other genes on your DNA template To make all of the complexes identical, you would like to arrest all transcriptional events at the same position on the DNA template before isolating the complex. How might you do this

Answer 3

To arrest the transcription events at same positions, a dinucleotide which is complementary to the first two nucleotides can be incorporated. This will not allow the incorporation of first nucleotide. Thus, we can obtain the small nucleotides each of same length which has been arrested at the same position. This can be arrested by adding only two or three instead of four (means one less than the required) rNTPs (ribonucleoside triphosphates) at the site where the transcription has to be arrested.

Question 4

For some time it was not clear whether lac repressor inhibits lac operon transcription by inhibiting the binding of RNA polymerase to its promoter or by allowing transcription initiation but blocking elongation past the site of bound repressor. How might you distinguish between these possibilities

Question 5

A lac operon containing one mutation was cloned into a plasmid, which was introduced by transformation into a bacterium containing a wild-type lac operon. The three genes of the chromosomal operon were rendered noninducible in the presence of the plasmid.
(a) What kind of mutation in the plasmid operon could have this
effect
(b) Suppose the result of transformation was to cause the three plasmid lac genes to be expressed constitutively, at a high level. What type of plasmid gene mutation could have this result

Question 6

Several new genes in the SOS regulon were identified by an ingenious use of "Mud" phages. These are derivatives of phage Mu that have a promoterless -galactosidase gene inserted at a particular point in this phage genome. How might these phages be used to identify genes whose expression is turned on after ultraviolet irradiation of bacteria

Question 7

Partial diploid forms of E. coli were created, each of which contained a complete lactose operon at its normal chromosomal site and the regulatory sequences only ( i, p, o )on a plasmid. Predict the effect of each mutation upon the activity of -galactosidase before and after the addition of inducer. Use , + or + + to indicate approximate activity levels.

Question 8

What type of mutation of the lac repressor might be both constitutive and trans -dominant

Question 9

It has been proposed that thiogalactoside transacetylase (LacA in the lactose operon) plays a role in detoxification-ridding the cell of potentially toxic -galactosides by acetylating them to facilitate their diffusion out of the cell. How might you test this proposal

Question 10

Consider Figure 29.34. Note that accumulation of EF-G and the subunit of RNA polymerase are subject to translational repression by ribosomal proteins S7 and S4, respectively. This would seem to be disadvantageous to the organism because S4, S7, and S8 binding to ribosomal mRNAs are mechanisms to control ribosome assembly. Why might this phenomenon not be disadvantageous

Question 11

Riboswitches are generally considered to have been discovered in about 2002. But a comparable regulatory process was described much earlier, when Nomura et al. ([1980] Proc. Natl. Acad. Sci. USA 77:7084) described the regulation of ribosomal protein synthesis carried out by binding of ribosomal proteins to their own mRNAs (Figure 29.35). Was the riboswitch actually discovered years earlier Discuss similarities and differences in control of ribosomal protein synthesis and riboswitch regulation as discussed in this chapter.

Question 12

Describe experimental evidence that would indicate that most or nearly all of the DNA sequences in a mammalian genome are transcribed.