Regulation. 1. Short term control 8-1

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1 Regulation Several aspects of regulation have been alluded to or described in detail as we have progressed through the various sections of the course. These include: (a) compartmentation: This was not described overtly as a control mechanism, but is implicit in the situation of β-oxidation of fatty acids (degradation) occuring inside the mitochondria and synthesis occuring in the cytoplasm. In this way, if one pathway is turned on, there is no competition from the other pathway operating in the opposite direction. (b) hormonal: The effect of hormones was mentioned in a few sections, but the main contact was in glycogen metabolism where adrenalin was shown to activate protein kinase in the complex process affecting glycogen synthesis and degradation. (c) feedback inhibition and activation: There were many examples of this ranging from energy metabolism to pyrimidine synthesis. This is often referred to as short term control (see below), because the inhibition or activation can occur as fast as an effector molecule can bind to a protein. (d) protein modification: This is best illustrated by the role of protein kinase and protein phosphatase in glycogen metabolism. This is also an example of post-translational control Other types of control occur at the level of RNA and protein synthesis (transcriptional and translational controls). Of these, transcriptional control has been found to be more common. These are often referred to as long term control (see below) because the response time is longer requiring either the synthesis or degradation of a protein. (e) control of RNA levels: As noted this control occurs at the level of either transcription or translation and is referred to as long term control. 1. Short term control 8-1 A B C In a linear pathway, the end product acts at an early step, shutting down the whole pathway and saving energy. Pi aspartate + carbamoyl phosphate aspartate transcarbamoylase feedback inhibition by an end product of the pathway N-carbamoyl apartate CTP

2 8-2 Whereas control of a linear pathway is relatively straightforward, control of a branched pathway presents more of a challenge because of the existence of multiple end products ( and G below). A system is needed to allow both end products a role in regulating overall expression of the pathway and a number of systems have evolved that make this possible. A B C F G (a) multiple enzyme control: Multiple alleles of the same enzyme exist to catalyze a step early in the pathway that are affected differently by different inhibitors. This controls the overall pathway but additional sites of control are required in each branch after the branch point. I A B C II F G (b) sequential feedback: The end products inhibit a step after the branch which cause a build up of an intermediate which acts sequentially to inhibit a step early in the pathway. A B C F G (c) synergistic feedback: two end products work in concert to inhibit a step early in the pathway and together have a much larger effect than individually. Variations on this include cumulative (the effect of and G are additive) and concerted (neither has an effect individually). 95% 10% A B C 15% F G

3 We will return to this topic towards the end of the section for examples. One amino acid biosynthetic pathway will suffice to provide examples of all three types of feed back inhibition in a branched pathway. However, that same pathway provides an example of how long term control can modulate a pathway. So we will first consider long term or transcriptional control mechanisms and then look at the example. 2. Long term control: control of transcription By controlling transcription, the amount of mrna encoding a protein or enzyme is regulated which in turn affects the amount of protein synthesized. Presumably one of the reasons that transcriptional controls are so common is that it provides for greater energy efficiency in stopping both RNA and protein synthesis. Two examples will be provided to illustrate transcriptional control. The first is in the catabolic (degradative for energy production) lac operon and the second is the anabolic (biosynthetic) trp operon. The differences inherent in the two operons are a reflection of the metabolic roles of the operons. (a) lac operon - negative and positive control of a catabolic operon An operon is a grouping of genes, the products of which have a common metabolic purpose. In the case of the lac operon, that purpose is the metabolism of lactose (galactose-β1,4-glucose) or milk sugar. The operon encodes a transport protein (lactose permease) to transport lactose into the cell and a cleavage enzyme (β-galactosidase). There is also a third gene encoding β-galactoside transacetylase presumably required for detoxification, but not important to this discussion. The key enzyme for this discussion is β-galactosidase which catalyzes two reversible reactions: 8-3 lactose (gal-β1,4-glu galactose + glucose allolactose (gal-β1,6-glu) The synthesis of β-galactosidase mrna is affected by the carbon sources present in the growth medium and changing them changes the expression. β-galactosidase mrna succinate + lactose + glucose (with lactose) repressed induced catabolite repressed Time

4 8-4 The rationale explaining the expression pattern is that the mrna encoding β-galactosidase is made only when there is a substrate (lactose) present for it to act on and only in the absence of a better substrate (glucose). (a) repressed: With succinate as carbon source, an enzyme to break lactose down is not needed and it would be a waste of energy to produce it. Synthesis of β-galactosidase mrna is repressed. (b) induced: With lactose added to the medium, breaking down lactose to galactose and glucose will produce a better substrate than succinate, so the synthesis of β-galactosidase mrna is induced. (c) catabolite repressed: When glucose is added to the medium, it is a better substrate than lactose because it is no longer necessary to break lactose down to produce glucose. The synthesis of β-galactosidase mrna is no longer needed and its synthesis is repressed by the catabolite glucose, hence catalabolite repression. These effects are all the result of specific effector proteins (a repressor and an activator) interacting with specific NA sequences in the control region (operator/promoter) of the NA of the lac operon. p i t o / p z y a t transcription mrna mrna translation repressor β-galactosidase permease transacetylase Regulatory "genes" i encodes the repressor protein o = operator or repressor binding site p = promoter t = terminator Structural genes z encodes β-galactosidase y encodes lactose permease a encodes β-galactoside transacetylase One additional protein that is required for the control of expression of the lac operon is encoded by a gene crp that is unlinked to the lac operon (is situated elsewhere on the chromosome). crp encodes a transcription activator protein variously know as catabolite gene activator protein (CAP) or camp receptor protein (CRP). ither name is acceptable although we will use CAP in these notes.

5 8-5 (i) repressed state: growth on succinate i o / p RNA-P + CAPcAMP CAP + camp The repressor binds to the operator which overlaps the promoter and prevents RNA polymerase + CAP from binding. active repressor (can bind to o) (RNA-P = RNA polymerase) (ii) induced state: lactose added to the growth medium CAP-cAMP facilitates RNA-P binding to the promoter. In so doing it activates transcription + + i o / p RNA-P CAPcAMP CAP camp mrna inactive repressor (cannot bind to o) allolactose Where does the allolactose come from? In fact, the operon is "leaky" and even when repressed there is a small amount of expression resulting in a small amount of lactose permease and β- galactosidase to bring lactose into the cell and convert it into allolactose. The increase in mrna levels can be as much as 1000 times. Binding of allolactose to the repressor protein causes a change in conformation that prevents the repressor from binding to the operator. In fact the repressor exhibits allosteric properties and we can consider it to exist in an equlibrium between the R (active) and T (inactive) states with allolactose displacing the equilibrium towards the T state. T R

6 8-6 (iii) catabolite repressed state: glucose added to the growth medium (lactose is still present) glucose causes camp levels to drop reducing the amount of CAP-cAMP o / p i + RNA-P CAP mrna (reduced to ~1/3 or less of fully induced) allolactose inactive repressor (cannot bind to operator) In fact, the term catabolite repression might actually be considered a misnomer because the process involves an activator protein (CAP) that becomes less active rather than a repressor protein. The lac repressor protein remains inactive as well because of the presence of lactose/allolactose. The lac operon is regulated by both negative (repression) and positive (activation) mechanisms. (b) trp operon - negative control in an anabolic (biosynthetic) operon Lec #24 Tryptophan is produced from intermediates in the pentose phosphate pathway taken through a very complex series of about 15 reactions. The final six steps are carried out by enzymes encoded in the trp operon. The mode of control here is conceptually quite different, in fact the opposite, from that in a catabolic operon. A catabolic operon is turned ON only when the substrate of the operon is present in order that energy is not wasted to make mrna and enzymes that are not needed. An anabolic operon is turned OFF when the product of the operon enzymes is present, in order that energy is not wasted to make mrna and enzymes that are not needed. glucose + tryptophan trp operon mrna induced repressed Time

7 8-7 (i) induced state: growth on glucose R o / p RNA-P C B A mrna inactive repressor (cannot bind to operator) (ii) repressed state: with tryptophan added R o / p RNA-P C B A tryptophan (a corepressor) active repressor-corepressor complex

8 3. xample of short and long term control in one pathway 8-8 The following example builds on the trp operon to provide not only an illustration of the three different types of short term control in a branched pathway but also another example of long term or transcriptional control. The mode of repressor action in the transcriptional control is exactly analogous to that in the trp operon in that amino acids act as co-repressors to turn off expression of certain genes. You will find the details for this pathway starting on page 6-8. The control will be broken into two separate stages: short term and long term. (a) short term: feedback inhibition multiple aspartate kinases aspartate I II III aspartyl-p aspartate semialdehyde multiple homoserine II I dehydrogenases homoserine lysine synergistic homoserine-p methionine threonine sequential α-ketobutyrate isoleucine

9 8-9 (b) long term: transcriptional control aspartate I II III aspartyl-p aspartate semialdehyde II I homoserine lysine homoserine-p methionine threonine α-ketobutyrate isoleucine Control occurs in two operons: 1. The genes encoding aspartyl kinase I and homoserine dehydrogenase II are part of the same operon controlled by threonine and isoleucine as corepressors. 2. The genes encoding aspartyl kinase II and homoserine dehydrogenase I are part of another operon controlled by methionine as a corepressor.

10 4. Summary Short term control - feedback inhibition in a linear and branched pathways 2. Long term control - control of transcription (a) lac operon - repressor (negative control) and CAP-cAMP (poisitive control) (b) trp operon - repressor and corepressor 3. xample of control mechanisms in the aspartate family of amino acids.

11 glycogen UPG Integration of Pathways - Overview glc ribulose-5p glc-1p glc-6p ribose-5p PRPP 8-11 CO 2 frc-6p IMP AMP GMP frc-1,6bisp ATP datp GTP dgtp Ga-3P HA-P dctp 1,3-bisPGA 3-PGA ser gly triglycerides phospholipids CTP UTP dttp 2-PGA ala thr ile thr UMP asp PP val pyruvate CO 2 AcCoA fatty acids ornithine citrulline OAA citrate simple lipids urea arg arg-succ malate fum glyoxalate isocitrate α-kg CO 2 NH 3 N 2 tyr phe AP + Pi FAH 2 NAH succ ATP succcoa CO 2 val NH 3 glu gln FA NA +