Biology 331, Genetics, 11/27/2017

1) Remember the fundamental concepts of Lac Operon regulation from Introductory Biology? Here’s a review:

The lac operon contains 3 structural genes that encode 3 proteins involved in utilization of lactose, a disaccharide, as an energy source. The operon is transcribed into a polycistronic mRNA that is translated into the three proteins.
Q1: What are the necessary elements that must be present in the polycistronic mRNA that allows it to encode 3 separate proteins?

A regulatory region 5’ to the transcription start site contains a PROMOTER, an OPERATOR, and a CAP binding site.
Q2: What proteins bind to each of the above DNA sequences?

Lactose serves as an inducer of lac operon transcription by binding to an allosteric site on the LAC REPRESSOR protein. Binding of lactose elicits a conformational change in the repressor, causing it to lose affinity for the Operator DNA sequence that lies between the Promoter and the transcription start site. RNA POLYMERASE binds to the Promoter, but cannot initiate transcription if Repressor is bound to the Operator.
Q3: describe the transcription status of the operon in presence and absence of lactose

The Catbolite Activator Protein (CAP) senses the types of sugars available to the cell by binding cAMP, whose concentration is INVERSELY correlated with GLUCOSE concentration (the higher the [glucose], the less cAMP is present). When cAMP is high, the allosteric binding site on CAP is occupied by cAMP, and the CAP has high affinity for the CAP binding site. This renders the lac operon (and other catabolic operons) available for RNA polymerase binding to the Promoter.

Here is a growth curve obtained with E. coli over time. Cells started at low density in medium containing both lactose and glucose.

Discuss and explain with your group the biphasic nature of the above growth curve. What is happening?

The following chart begins by summarizing some of the data of Jacob and Monod that led them to the model of lac operon regulation, winning them the Nobel Prize for the discovery of mechanisms of gene regulation. IPTG is a non-hydrolyzable analog of the disaccharide, lactose. + and – in the IPTG columns refer to the presence or absence of IPTG in the culture medium. The superscripted + and – and S and c in the genotypes column refer to alleles of either the structural genes (Z or Y) or the regulatory I gene (repressor) or the Operator sequence. + means wild type, - means a null mutation, c means “constitutive” (always on) and S means “super” (always off). The + and – in the B-gal and Permasesupercolumns refer to the presence or absence of the lac Z or lac Y gene products produced by two of the structural genes of the lac operon.

Your job is to fill in the rest of the chart for the merodiploid genotypes in last 6 rows of the chart. DM will explain the nature of merodiploids in class.