Download Regulation of Gene Expression

BS/LBS159H,
SS’04
Regulation of Gene Expression
Turning Genes On and Off
Introduction
Genes are not distributed randomly within a genome, nor are they continually
expressed. Instead, cells organize genes and regulate their transcription in ways to
conserve energy and respond efficiently to environmental conditions. This week you
will be introduced to some of the ways that genes are organized within a bacterial
genome and some strategies that bacteria use to regulate their expression. In the
laboratory, you will manipulate the expression of Green Fluorescent Protein (GFP) in the
GFP-transformed E. coli you created in last week’s cloning exercise.
Learning objectives
Conceptual
Organisms have devised ways of organizing genes within their genome to control their
expression efficiently.
Cells can sense their environment and change in response to it by turning on or off
genes through the use of regulatory proteins.
Practical
By this time in the class you have already been exposed to the plating and sterile
techniques that we will be applying in this exercise.
Underlying
Science
E. coli has an estimated 4403 protein-coding genes—only a fraction of which are
expressed at any one time. This fact seems more obvious in the case of more complex
multicellular organisms like humans. The human genome encodes 50 thousand or more
genes, and it is obvious from our complex development, various organs, tissue and cell
types that different cells in our body express different genes. Experiments have
proven this. For example, liver cells express a different set of genes than the cells of
your brain (or any other organ for that matter). This is understandable in that the liver
has a wholly different function than the brain.
In the case of single-celled organisms, this is not as obvious because they are not
composed of different tissues. However, bacterial cells have to deal with changing
environmental conditions and, while expressing all of their genes at once would
accomplish this, it would require an unnecessarily excessive expenditure of energy.
Alternatively, if an organism could turn its genes on and off only when they are needed,
in response to the environmental conditions in which the cell finds itself, it could utilize
energy and available resources in the most economical manner and be more
competitive in its habitat. Advances in biotechnology and molecular techniques has
recently spawned a new field in biology
Bacterial cells accomplish this at a number of levels.
First of all, genes within a bacterial genome are not distributed randomly but
instead are organized into operons (see below) that allow for efficient
transcription of related genes (genes associated with a common process or
involved in a multi-step pathway).
Second, genomes include genes for regulatory proteins that are used to turn
gene expression on and off (regulate transcription) as needed.
Thirdly, translation can be regulated.
Finally, cells can control the activity of enzymes already present in the cell.
Some genes DO need to be expressed all the time. Genes that encode proteins of
basic essential functions such as DNA replication, RNA transcription or protein
translation are called housekeeping genes. Typically housekeeping genes are always
on. This is called constitutive expression.
Not all genes are transcribed individually. Genes associated with a particular process or
pathway (say the enzymes needed in the multi-step process of synthesizing the amino
acid tryptophane) will often be located adjacent to each other in the genome and be
transcribed on a single piece of mRNA. Individual proteins will then be translated off of
that mRNA. Transcription is carried out by the enzyme RNA polymerase. RNA
polymerase does not just randomly attach to the chromosome but recognizes specific
binding sites called promoters. ..
So a promoter is a region of the genome that RNA polymerase attaches to initiate
transcription and an operon is a group of functional genes all transcribed from a single
promoter.
This is a logical arrangement in that ALL of the proteins needed for a process will be
made available when they are needed. It would be a waste of energy and resources to
synthesize the first enzyme in some pathway if the next enzyme in the pathway isn’t
available to continue the process. But this also allows the bacterial cell to turn an
entire process on and off in response to environmental conditions by controlling the
ability of RNA polymerase to bind the associated promoter site. It does this by using
regulatory genes that encode for proteins that either inhibit RNA polymerase binding
(repressors) OR actually promote RNA polymerase binding (inducers) that act upon
the promoter site in response to environmental ques.
Different operons employ different strategies and combinations of induction and
repression. You will be introduced to some of these in lecture. One operon, the
arabinose operon, uses a regulatory protein, araC protein, that can act as EITHER
a repressor OR an inducer of the arabinose operon promoter in response to whether
arabinose is available to the cell or not.
The araC protein is important to this week’s exercise because the the pGLO plasmid
you cloned last week (and will be using this week) was engineered to contain the GFP
gene under control of an arabinose operon operator and the gene for the regulatory,
araC protein.
This means that we cloned the GFP gene into E. coli but it will only be expressed if
arabinose is made available to the cells. Therefore we can turn the GFP on and off at
will by simply supplying or starving the cells of arabonose (arabinose is a sugar that the
bacterium can use as a carbon and energy source).
Protocol
Variables and Controls
Independent variable
Presence or absence of arabinose
Dependent variable
Expression of GFP
Control variables
Transformed strain of E. coli, base medium of the agar, incubation
temperature, etc.
Materials
Your LBamp plate and your LBamp/arab plates of E. coli transformed with the pGLO
plasmid from last week
One LBamp plate
One LBamp/arab plate
Bunsen burner and inoculating loop
Procedure
1) Pick a colony from your LBamp plate and streak a LBamp/arab plate.
Is the GFP gene turned on or off on your LBamp inoculum plate (i.e. does it
fluoresce or not?) Do you expect it to be expressed on the LBamp/arab plate
after you incubate it.
2) Pick a colony from your LBamp/arab plate and streak a LBamp plate.
Is the GFP gene turned on or off on your LBamp/arab inoculum plate (i.e.
does it fluoresce or not?) Do you expect it to be expressed on the LBamp
plate after you incubate it.
3) Incubate your plates at 37oC for 24 to 48 hours and observe them on the UV
transilluminator.
4) Bring in an IBM-formatted floppy and use the digital camera to record your
results.