Transcriptional Control: Bacteria

Sections

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TRANSCRIPTIONAL CONTROL-BACTERIA

Operon

Cluster of genes that are transcribed into one long mRNA allowing the genes of a single pathway to be controlled with a single on/off switch

Operator

Segment of DNA that acts as a switch to control access of RNA polymerase to the gene

Activator

Protein that binds to DNA and stimulates gene transcription

Negative regulation

Bound repressor protein blocks transcription

Positive regulation

Bound activator protein promotes transcription

LAC OPERON

• Set of genes that code for proteins necessary for the bacterium to use the sugar lactose as an energy source

Structure

• 3 genes: lacZ (beta-galactosidase), lacY (lactose permease) and lacA (galactoside acetyltransferase)
• Promoter region:

1. CAP-binding site
2. Operator

• lacI gene – prior to CAP-binding site; codes for repressor protein; under control of a different promoter

High glucose, no lactose

• CAP-binding site empty (inactive catabolite activator protein due to low cAMP levels)
• Repressor is bound to operator (no allolactose present to inactivate repressor)
• No transcription

No glucose, no lactose

• CAP is bound to CAP-binding site (low glucose means high levels of cAMP)
• Repressor is bound to operator
• No transcription

High glucose, lactose available

• Cap-binding site empty
• Operator is empty (allolactose present inactivates repressor protein)
• Low-level transcription

No glucose, lactose available

• CAP is bound to CAP-binding site
• Operator is empty
• High levels of transcription

Overview of Bacterial Chromosome Replication

Overview of Horizontal DNA transfer

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• Here we will learn about transcriptional control of gene expression in bacteria. We discuss the transcriptional control of gene expression in eukaryotes elsewhere.

• First, start a table to understand some key terminology.

• Denote that an operon is a cluster of genes that are transcribed into one long mRNA.
  • Genes coding for enzymes in the same pathway are grouped together in the chromosome.

• Denote that this allows the genes of a single pathway to be controlled with a single on/off switch.

• Denote that an operator is a segment of DNA that acts as the switch to control access of RNA polymerase to the gene.

• Denote that a repressor is a protein that binds the operator and blocks attachment of RNA polymerase.

• Finally, denote that an activator is a protein that binds to DNA and stimulates gene transcription.

As an example to better understand bacterial transcriptional regulation, let's look at the lac operon found in E. coli.

• Write that it is a set of genes that code for proteins necessary for the bacterium to use the sugar lactose as an energy source.

• If lactose isn't present, why produce these enzymes, at all?
  • If glucose is present, the bacterium would preferentially use it over lactose.
  • So, control of the lac operon must take the levels of lactose and glucose into account.

We'll start with the organization of the lac operon.

• Draw a stretch of DNA.

• Indicate three sections of the DNA as the three genes, lacZ, lacY, and lacA.
  • lacZ codes for beta-galactosidase.
  • lacY codes for lactose permease.
  • lacA codes for galactoside acetyltransferase.

• Label the section of DNA before the genes the promoter.

• Within the promoter label the operator and the CAP-binding site.
  • Some texts only consider the region of DNA between the CAP-binding site and the operator the promoter.

• Prior to the CAP-binding site, label a section of the DNA as the gene for lacI.
  • lacI codes for a repressor protein that is part of the lac operon control mechanism.
  • It is under the control of a different promoter.

• Draw the RNA polymerase bound to the DNA and indicate the direction it travels during transcription.

Now let's explore how the levels of glucose and lactose affect gene transcription levels.

• We'll look at four situations:
  • High glucose with no lactose;
  • No glucose with no lactose;
  • High glucose with lactose available; and
  • Low glucose with lactose available.

So what happens in the situation where the bacterium has access to glucose but not lactose?

• Draw the lac operon DNA.

• Bound to the operator, draw the lac repressor protein.
  • The protein has a binding site that recognizes an isoform of lactose and is inactivated when the isoform is bound.

• Indicate that the protein is active.
  • When active and bound to the operator, the repressor blocks transcription.

• Draw the inactive catabolite activator protein (CAP) which has a cyclic AMP (cAMP) binding site.
  • Cyclic AMP concentrations increase when glucose is scarce.

• Write that the CAP-binding site is empty and the repressor is bound to the operator.

• Draw the RNA polymerase and indicate that it is unable to bind the DNA.

• Write that there is no transcription in this situation.

• Write that this is an example of negative regulation where a bound repressor protein blocks transcription.

How about when the bacterium has doesn't have access to either glucose or lactose?

• Draw the lac operon DNA.

• Because there is no lactose present, draw the activated repressor protein bound to the operator.

• Because there is no glucose present, draw the CAP and cAMP bound to the CAP-binding site.

• Indicate that CAP is active because cAMP is bound to it.

• Write that CAP is bound at the CAP-binding site.

• Write that the repressor is bound to the operator.

• Draw the RNA polymerase and indicate that it is unable to bind the DNA.

• Write that there is no transcription in this situation.

What happens when glucose and lactose are both available?

• Draw the lac operon DNA.

• Because there is glucose, there is no cAMP available to bind CAP.

• Draw the inactive CAP.

• Because lactose is present, allolactose (which is an isoform of lactose) is available to bind the repressor protein and inactivate it.

• Draw the inactive repressor protein with allolactose bound to it.

• Write that the CAP-binding site is empty.

• Write that the operator is empty.

• Draw RNA polymerase bound to the DNA and indicate that it is able to transcribe.

• Write that in this situation, there is low-level transcription of the lac operon genes.

Finally, what happens when there are low levels of glucose and lactose is present?

• Draw the lac operon DNA.

• Because there are low levels of glucose, cAMP levels are increased.

• Draw CAP bound to the CAP-binding site.

• Draw cAMP bound to CAP and indicate that CAP is active.

• With lactose available, allolactose binds to the repressor protein and inactivates it.

• Draw the inactive repressor protein with allolactose bound to it.

• Write that CAP is bound at the CAP-binding site.

• Write that operator is empty.

• Draw the RNA polymerase bound to the DNA.

• Indicate that transcription is able to proceed.

• CAP is an example of a positive regulator.

• Write that positive regulation occurs when a bound activator protein promotes transcription.

UNIT CITATIONS:

1. Campbell, N. A. & Reece, J. B. Biology, 7th ed. (Pearson Benjamin Cummings, 2005).

2. Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K. & Walter, P. Molecular Biology of the Cell, 5th ed. (Garland Science, 2008).

3. Alberts, B., Bray, D., Hopkin, K., Johnson, A., Lewis, J., Raff, M., Roberts, K. & Walter, P. Essential Cell Biology, 3rd ed. (Garland Science, 2010).

4. Lodish, H., Berk, A., Kaiser, C. A., Krieger, M., Scott, M. P., Bretscher, A., Ploegh, H. & Matsudaira, P. Molecular Cell Biology, 6th ed. (W. H. Freeman and Company, 2008).

5. Perdew, G. H., Vanden Heuvel, J. P. & Peters, J. M. Regulation of Gene Expression: Molecular Mechanisms. (Humana Press, 2006).

6. Hames, D. & Hooper, N. Instant Notes in Biochemistry. (Taylor & Francis, 2004).

7. Krebs, J. E., Lewin, B., Goldstein, E. S. & Kilpatrick, S. T. Lewin's Essential Genes. (Jones & Bartlett Publishers, 2013).

8. Snape, A., Papachristodoulou, D., Elliott, W. H. & Elliott, D. C. Biochemistry and Molecular Biology. (OUP Oxford, 2014).

9. Lewin, B., Krebs, J., Kilpatrick, S. T. & Goldstein, E. S. Lewin's GENES X, Volume 10. (Jones & Bartlett Learning, 2011).

10. Wood, B. J. B. & Warner, P. J. Genetics of Lactic Acid Bacteria. (Springer Science & Business Media, 2012).