The bacterium Escherichia coli has about 3000 genes.. For many (probably most) of these genes, there is some kind of
regulation of their expression (so they are "on" when they should be on, and
"off" when they should be off). In bacteria, regulation of gene expression is
mainly for responding to changing environmental (chemical and physical) conditions
that the bacterium finds itself in. In large multicellular organisms, regulation
of gene expression is mainly for the embryological development of the organism,
for the specificity of function of the various organs and tissues, and for maintaining
the appropriate levels of the many enzymes and other proteins that the cells
of the organism need.
In E.coli, the lac operon is a group of three genes that code for the enzymes needed for lactose (a sugar) utilization by the cell. In an E. coli cell growing in a growth medium containing glucose as the
only carbon source, the lac operon is "off" (not being
transcribed). If we add lactose to the growth medium, the lac operon remains
"off", with the cell continuing to utilize glucose. When the glucose is gone, however,
the lac operon turns "on" and synthesizes the enzymes needed for the
cell to use the lactose as a carbon source. These "molecular decisions"
get made in an approximately 100 base pair region in the DNA called the lac
control region, and today we will look at the details of how it works.
1. What is the distinction between the terms "negative
regulation" and "positive regulation"? (covered in your text on pages 446-448).
"Negative regulation" involves a regulatory protein binding to the
DNA somewhere near a promoter to PREVENT RNA polymerase from binding and starting
transcription.
"Positive regulation" involves a regulatory protein binding to the
DNA somewhere near a promoter to ALLOW RNA polymerase to bind and start transcription.
2. In general terms, how do BOTH of these types of regulation act together
to provide a very effective "on-off" switch for the expression of the group
of 3 genes (z , y, a) that get expressed together to give the enzymes
for lactose metabolism in E. coli?
Figure 11.6 "top one" illustrates that repressor binding to the DNA
near the promoter PREVENTS expression. This occurs when there is no lactose
present; when lactose is present, it prevents repressor from binding to the
DNA.
Figure 11.6 "third one" illustrates that CRP-cAMP binding to the DNA
near the promoter is NEEDED for expression. This allows expression to occur
only when there is no glucose present, and thus cAMP levels are high {cellular
biochemistry keeps the intracellular levels of glucose and cAMP inversely correlated}.
Figure 11.6 "second one" shows that there is no expression when both
proteins are bound. This situation occurs when a cell is growing in a carbon
source other than either glucose or lactose.
Figure 11.6 "fourth one" shows that there is no expression when neither
protein is bound. This situation occurs when a cell is growing in a medium containing
both glucose and lactose.
3. In specific terms, how does this 100 base pair control region work?
Figure 11.7 shows the base pair sequence in the E. coli genome in the immediate vicinity of the transcription start site of the lac operon. The sites to which RNA polymerase, repressor, and CRP protein bind are shown. A closer look shows that the region just to the left of the "protected by CRP" site has a base pair sequence very similar to the already-known repressor binding site (the "operator") at the right end of the promoter. This sequence similarity suggests that perhaps repressor protein, which is a tetramer of four identical polypeptide chains (assembled into a structure with mirror-image symmetry), may actually bind to BOTH of these sites. Experiments in 1996 showed that this is indeed the case.
Figure 11.8 shows how the repressor protein binding to these two regions along
the DNA causes the DNA to "loop". If the DNA is in this configuration, RNA polymerase
cannot bind effectively to the promoter, and thus transcription of the lac operon does not occur. If the DNA is NOT in this configuration, RNA polymerase
CAN bind to the promoter IF CRP-cAMP is bound just to the left of the promoter.
It is important to understand Figures 11.7 and 11.8 clearly!
Problem S-8: "lac control region configuration":
For the four growth situations listed below, make
clear drawings of the configuration of the approximately 100 base pair
lac control region, and show any and all proteins that are bound, or can
bind, to it. For each case, are RNA polymerase enzyme molecules able to bind
to the promoter and start transcription? Why or why not?
1. An E. coli cell growing in a glucose medium (no lactose in medium).
2. Add lactose, so now the cell is growing in a medium containing glucose and
lactose.
3. After a while, the glucose is gone, so now the only carbon source is lactose.
4. Later, the lactose is gone, and you add a different carbon source such as
the sugar fucose.