Biology 210
GENETICS
25 March, 1998
Chapter 11a (pages 459-470) Control of Gene Expression in Bacteria
1. Basic Control Circuts

2. Discovery of the lac System:
Negative control

3. Catabolite Repression of the lac Operon: Positive control

For a few links about gene regulation, see the page from the biology place (check my email for descriptions on how to log in). Also, you might want to look at the transcription & translation sites, as well as listen to the "Ribosome song" (sung to the tune of "Roll Over Beethoven" by Chuck Berry).

1. Basic Control Circuts - BACKGROUND
A. Bacterial chromatin

The Problem Stated:
DNA Compaction
E.coli has a problem. The DNA must be compacted more than a thousand fold, yet it still needs to be avaliable to be transcribed.

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This is a famous electron micrograph of an E.coli cell. The bacterium has been carefully lysed, then all the proteins were removed, and the remnant of the cell was spread on an EM grid to reveal all of its DNA. This is the same as Figure 6.3 (page 227 in Hartl & Jones, 1998).




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The length of a typical bacterial operon (usually about 3 genes), is about as long as the entire bacterial cell, if it is stretched out in its B-DNA double helical conformation!

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<-----length of bacteria------>


Remember from the lecture on DNA supercoiling, about half of the supercoils are constrained. RNA polymerase is responsible for constraining roughly HALF of these supercoils.








B. REGULATION
of gene expression in bacteria

Obviously, not all of the genes in a bacteria will be expressed at the same time. Even in some of the smallest bacteria, there are about 500 different genes; it would be wasteful for the bacteria to constantly express all of these genes.

link to lecture on Genomics

Table 1. Comparison of genomes of various organisms
ORGANISM	% coding	Size of genome (bp)	number of genes
Escherichia coli	90%	4,639,221 bp	4288
Mycoplasma genitalium	88%	580,073 bp	468
Haemophilus influenzae	86%	2,087,778 bp	1,662
Methanococcus jannaschii	85%	1,660,000 bp	1,997
Synechocystis sp. (PCC 6803)	80%	3,570,000 bp	3,168
Saccharomyces cerevisiae	~75%	13,000,000 bp	6,275
Humans	~2%	3,000,000,000 bp	70,000 (?)

E.coli is the best characterised organism.


some numbers:

• There are 4288 genes in Escherichia coli.


• Roughly 2600 of these genes have been found under standard laboratory growth conditions.


• About 2100 spots can be seen on 2-D protein gels.


• Only about 350 proteins exist at concentrations of > 100 copies per cell. (These make up 90% of the total protein in E.coli!)


• Most (>80%) of the proteins are present in very low amounts (less than 100 copies per cell).

These (e.g., the majority of the) genes are likely to be expressed transiently, in small amounts during DNA replication, and then remain silent (unexpressed) until the next round of DNA synthesis.




The expression of only a small number of genes is REGULATED at all. There are many different types of regulation. Most genes are regulated at the point of transcription initiation, although transcriptional elongation and termination, mRNA stability, translation, and protein modification and stability are all possible points of regulation.

Gene regulation can occur at any place along the flow of information from DNA to RNA to protein:

Compare this list to the 6 points from Hartl & Jones, page 460. Essentially their list is the same - I've left off DNA rearrangements (and also DNA amplification: an easy way to get more protein is to have more copies of its DNA!), and the last three of the points on page 460 in your text (translational control, mRNA stability, and post-translational control) are all covered in my section on "post-transcriptional control". Most of these events, although important, play a secondary role in regulation of gene expression.

1. Regulation by DNA Replication (default)



2. Transcriptional Regulation by different s-factors.



E.coli RNA Polymerase subunits

Gene	Mass KDa	Use	-35 Sequence	separation	-10 Sequence
rpoA	40	a subunit	-	-	-
rpoB	155	b subnit	-	-	-
rpoC	160	b' subunit	-	-	-
rpoD	70	s70 General	TTGACA	16-18 bp	TATAAT
rpoN	54	s54 Nitrogen	CTGGNA	6 bp	TTGCA
rpoS	38	s38 Stationary	not known	not known	not known
rpoH	32	s32 Heat shock	CCCTTGAA	13-15 bp	CCCGATNT
fliA	28	s28 Flagellar	CTAAA	15 bp	GCCGATAA
rpoE	24	s24 High temp. heat shock	not known	not known	not known




• s⁷⁰ - RpoD is the “normal” s-factor
• s⁵⁴ - RpoN - Nitrogen response
• s³⁸ - RpoS - Stationary phase
• s³² - RpoH - Heat shock response
• s²⁸ - FliA - Flagellar genes regulation
• s²⁴ - RpoE - high temp. Heat shock




• There are about 1500 - 2000 copies of RNAP holoenzyme per cell (that is, a, b/b')


• For bacteria growing in "log phase":
• ~600 copies of RpoD (s⁷⁰)
• ~200 copies of RpoS (s³⁸)


• [RpoS] increases to ~600 copies per cell in stationary phase or osmotic shock.

Link back to lecture on Transcription (Friday, 20-Mar-98)




3. Negative Regulation of Gene Expression

By default, the gene is usually switched ON.
Binding of a REPRESSOR will switch the gene OFF.
This is most common form of regulation in BACTERIA
Often this is found as a form of AUTOREGULATION - where too much of the gene product inhibits further transcription - usually this is through binding to the upstream promoter control region. A good "classic" example of this is the E.coli lac operon (see below).





4. Positive control of Regulation

By default, the gene is usually switched OFF.


Binding of a ACTIVATOR will switch the gene ON.
(often transcriptional activators are called “transcription
factors”, which will bind and bend DNA upstream of the
promoter.)
This is most common form of regulation in EUKARYOTES

Some promoters are not very functional in the absence of a transcriptional activator protein(s). A well studied system is cAMP-CRP control of gene expression, and this system plays a crucial in Salmonella virulence. (see the lac example, below)



5. Alternative splicing of RNA
(almost exclusively for Eukaryotes)




6. Post-transcriptional regulation

termination of transcription
translation control
message stability
protein stability


The Lactose operon.

Most genes in bacteria are switched on by default....



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The product of the lac gene will cleave lactose sugar into galactose and glucose....

References

This lecture is based MAINLY on the following references:


Dorman, C.J., Genetics of Bacterial Virulence, (Oxford: Blackwell Scientific Publications, 1994).

Gralla, J.D. and Collado-Vides, J. "Organisation and function of transcription regulatory elements", In Escherichia coli and Salmonella typhimurium, cellular and molecular biology. F.C. Neidhardt, ed. (Washington, D.C.: ASM Press), pp. 1232-1245. (1996).

+Lewin, B. Part 4 - "Control of prokaryotic gene expression", In GENES V. (Oxford: Oxford University Press), pp. 375-524. (1993).

Neidhardt, F.C. and Umbarger, H.E. "Chemical composition of Escherichia coli", In Escherichia coli and Salmonella typhimurium, cellular and molecular biology. F.C. Neidhardt, ed. (Washington, D.C.: ASM Press), pp. 13-16.(1996).

Pettijohn, D.E. "The Nucleoid", In Escherichia coli and Salmonella typhimurium, cellular and molecular biology. F.C. Neidhardt, ed. (Washington, D.C.: ASM Press), pp. 158-166.(1996).

Ross, W., Gosink, K., Salomon, J., Igarashi, K., Zhou, C., Ishihama, A., Severinov, K., and Gourse, R. L. (1993). "A third recognition element in bacterial promoters: DNA binding by the alpha subunit of RNA polymerase". Science 262:1407-1413.

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Last modified on: 2 February, 2000 by Dave Ussery