Question

Areas of a chromosome that are actively being transcribed undergo a certain chemical transformation. What is it and what physical property of this transformation allows for efficient transcription?

Describe how the lac operon responds when E. coli goes from an environment of low/no lactose to an environment rich in lactose.  

What element of DNA does the sigma (?) subunit of RNA polymerase II interact with?

Answer 1

Areas of chromosomes that are actively being transcribed and under chemical transformation are DNA.

In calcium chloride transformation, the cells are prepared by chilling cells in the presence of Ca2+
(in CaCl2 solution), making the cell become permeable to plasmid DNA. The cells are incubated on ice with the DNA, and then briefly heat-shocked . This method works very well for circular plasmid DNA. Non-commercial preparations should normally give 10⁶ to 10⁷ transformants per microgram of plasmid; a poor preparation will be about 10⁴/?g or less, but a good preparation of competent cells can give up to ~10⁸ colonies per microgram of plasmid. Protocols, however, exist for making supercompetent cells that may yield a transformation efficiency of over 10⁹. The chemical method, however, usually does not work well for linear DNA, such as fragments of chromosomal DNA, probably because the cell's native exonuclease enzymes rapidly degrade linear DNA. In contrast, cells that are naturally competent are usually transformed more efficiently with linear DNA than with plasmid DNA.

The transformation efficiency using the CaCl2 method decreases with plasmid size, and electroporation therefore may be a more effective method for the uptake of large plasmid DNA.

Lac operon: The lac repressor is a protein that represses (inhibits) transcription of the lac operon, by binding to the operator,When lactose is not available, the lac repressor binds tightly to the operator, preventing transcription by RNA polymerase. However, when lactose is present, the lac repressor loses its ability to bind DNA.

Sigma factors have four main regions that are generally conserved:

N-terminus --------------------- C-terminus
             1.1    2    3    4

The regions are further subdivided

• Region 1.1 is found only in "primary sigma factors" (RpoD, RpoS in E.coli). It is involved in ensuring the sigma factor will only bind the promoter when it is complexed with the RNA polymerase.
• Region 2.4 recognizes and binds to the promoter ?10 element (called the "Pribnow box").
• Region 4.2 recognizes and binds to the promoter ?35 element.