D: Questions

Question 1

Allosteric proteins are an integral part of operon and regulon repression systems in prokaryotes. Explain the basic principle of allostery, and outline the main points of a prokaryotic gene regulatory system where allostery is used to effect control of gene transcription?

Answer 1

Allostery in the context of gene transcription: integrity of structure (shape) of DNA binding site on repressor-activator protein depends on binding of inducer molecules at separate site. Examples could range from lac or trp operons, maltose regulon or catabolite repression.

Question 2

Briefly outline the main points relating to feedback repression of the tryptophan option in E. coli?

Answer 2

A regulator codes for a normally inactive repressor protein (tryptophan is needed by the cell). If tryptophan becomes plentiful, it binds to the repressor, acting as a corepressor, and this allows the repressor to bind to the operator region of the promoter, preventing RNA polymerase from binding, and switching off transcription of the biosynthetic operon.

Question 3

If E. coli is growing in a medium that contains tryptophan, is the product of the trp R gene active or inactive?

Answer 3

Active

Question 4

What is the difference between enzyme regulation via repression and inhibition?

Answer 4

Repression refers to regulation at the level of transcription, while inhibition is at the level of enzyme activity.

Question 5

How does non-competitive inhibition control of an enzyme work in a metabolic pathway?

Answer 5

Non-competitive inhibition regulates enzyme activity. To prevent overproduction of the end-product in a metabolic pathway, the cell uses an allosteric enzyme to catalyze the first reaction. The end-product binds to an allosteric site on this enzyme, causing a change in the enzyme’s shape that alters the active site. This temporarily inhibits the enzyme’s activity, effectively controlling the pathway.

Question 6

Briefly, explain the difference between competitive and non-competitive inhibition? How does non-competitive inhibition control of an enzyme work in a metabolic pathway?

Answer 6

Both relate to control of enzyme activity at the post-translational level. For non-competitive inhibition, an enzyme (A) controlling an early step in a biochemical pathway has an active site plus a separate allosteric site. A build-up of end-product if the pathway results in the end-product binding to the allosteric site of A, changing the conformation of the active site, and so abolishing activity. In competitive inhibition, the end-product competes with the substrate of A for the active site.

Question 7

What is meant by the term ‘metabolic uncoupling’? Briefly, what is the relevance of this for biotechnology processes?

Answer 7

Metabolic uncoupling: homeostasis involves the active transport out of the cells of end-products, H+ and toxic compounds. Generation of acid end-products due to fermentative metabolism will lead to more ATP being directed away from biosynthesis towards homeostasis. An example is the metabolism of glucose to acetate in E. coli, which is undesirable and leads to the cell expending energy to exclude hydrogen ions.
The result is an undesirable lowering of biomass yields.

Question 8

Why is enzyme regulation important in biotechnology?

Answer 8

1. Production of many commercially important enzymes (such as α-amylase) is under inducible regulatory control. The cell environment (culture medium especially) can have a profound effect on enzyme synthesis.
2. A knowledge of inducing substances and the kinetics of induction is necessary to optimize process productivity.
3. Regulatory mutations can be used to eliminate the dependence of enzyme formation on an inducer (save time and cost of inducer addition)

Question 9

constitutive expression

Answer 9

enzyme systems that catalyse essential cellular metabolic reactions are switched on permanently (e.g. enzymes for glucose catabolism)

Question 10

operon

Answer 10

a regulatory protein that controls the transcription of a polycistronic mRNA – genes of related function

Question 11

regulon

Answer 11

a set of related genes controlled by the same regulatory protein, and they may be some distance from each other on the chromosome

Question 12

The lactose (lac) operon

Answer 12

inducible degradative system: common in the catabolism of carbon and energy source

Question 13

tryptophan biosynthesis

Answer 13

the regulator gene (trp R) codes for an inactive repressor protein which is therefore unable to bind to the operator region of the operon (and unable to block tryptophan synthesis)

RNA polymerase is able to bind to the promoter region of the operon & transcribes the five genes coding for the enzymes in the tryptophan biosynthetic pathway into mRNA

if tryptophan is plentiful, the organism switches off tryptophan biosynthesis

Question 14

tryptophan attenuation

Answer 14

‘fail-safe’ mechanism which acts at the junction of transcription and translation to down-regulate tryptophan biosynthesis (when tryptophan is available)

Question 15

maltose regulon function

Answer 15

controls the breakdown of the disaccharide maltose, to two glucose units

Question 16

Positive control of transcription (maltose)

Answer 16

control is effected by activators and these control genes that have a promoter to which RNA polymerase cannot bind. The promoter is beside a segment of DNA called the activator binding site. The genes needed for maltose catabolism are spread out in several operons, each of which possesses a binding site for maltose activator protein

Question 17

maltose regulon

Answer 17

maltose regulon comprises 10 genes distributed in 5 operons (producing 4 enzymes, 5 transport proteins and a periplasmic protein). The enzymes MalP, MalQ, MalS and MalZ catalyze the breakdown of maltose and maltodextrins (7-8 glucose residues) to produce glucose and glucose-6-phosphate. An activator protein (Mal T) controls genes that have a promoter to which RNA polymerase cannot bind, and in the absence of the inducer, maltose (and ATP), no transcription occurs. Binding of the inducer, maltose, induces a conformational change in the allosteric regulator. The regulator protein can now bind the activator binding site: this allows the RNA polymerase to recognise the promoter, and transcription takes place

Question 18

catabolite repression

Answer 18

if different carbohydrates are present in a medium under non- limiting substrate conditions, the most easily utilizable carbon source will be used preferentially (e.g. glucose)

Question 19

Catabolite repression by: Catabolite Activator Protein (CAP) or also known as cAMP Receptor Protein

Answer 19

Transcription of the catabolite genes by RNA polymerase can only occur if CAP is first bound to the CAP site on the DNA. CAP can only do this if it has first bound cyclic adenosine monophosphate (cAMP, acting as an effector molecule). In the presence of glucose, cAMP level is lowered and it is not available to bind to CAP

Question 20

competitive inhibition

Answer 20

the end-product of a pathway has a structure which is quite similar to the substrate of the first biochemical reaction, and so competes with it for binding to the active site (example: succinate dehydrogenase and malonate in the Krebs cycle)