Botany B Sc III Gene Regulation Shri Maruti

Botany B Sc III Gene Regulation Shri. Maruti A. Hake Botany

Intro • What causes gene products to be synthesized in some cells under some conditions, but not in others? A large part of research in molecular biology in aimed at trying to determine this. We are going to talk about a few basic systems, but a whole course could easily be devoted to the subject. • For most genes, the essential regulation point is transcription: whether the gene is transcribed or not. Regulation also occurs at other points: availability of the DNA to be transcribed at all, whether the m. RNA is translated, stability of the m. RNA, how quickly the protein is degraded, etc.

Gene Regulation in Prokaryotes • The first system of gene regulation that was understood was the lac operon in E. coli, worked out by Francois Jacob and Jacques Monod in 1962. Many other prokaryotic genes are regulated in a similar fashion, and the basic principles carry over into eukaryotes. • The lac operon codes for enzymes involved in the degradation of lactose. Lactose is a disaccharide that can be used as food in the absence of glucose. A lac- mutant is a chemoauxotroph that can’t use lactose.

Structure of the lac Operon • The lac operon consists of 3 protein-coding genes plus associated control regions. • The 3 genes are called z, y, and a. lac. Z codes for the enzyme beta-galactosidase, which splits lactose into glucose plus galactose. lac. Y codes for a “permease” protein that allows lactose to enter the cell, and lac. A codes for an enzyme that acetylates lactose. Together these three genes are called the “structural genes”. We will mainly focus on lac. Z. • All 3 genes of the lac operon are transcribed on the same messenger RNA. Ribosomes translate the 3 proteins independently. This is a feature of prokaryotes that is only very rarely seen in eukaryotes, where 1 gene per m. RNA is the rule.

Control Regions • Near the lac operon is another gene, called lac. I, or just “i”. It codes for the lac repressor protein, which plays an essential role in lac operon control. The lac repressor gene is expressed “constitutively”, meaning that it is always on (but at a low level). It is a completely separate gene, producing a different m. RNA than the lac operon. • Just upstream from the transcription start point in the lac operon are two regions called the operator (o) and the promoter (p). Neither region codes for protein: they act as binding sites on the DNA for important proteins. • The promoter is the site where RNA polymerase binds to start transcription. Promoters are found upstream from all protein-coding genes. • The operator is where the actual control occurs.

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Control • The lac repressor protein (made by lac. I) has 2 states: it can either bind to lactose (technically, to a lactose derivative called allolactose) or it can bind to the operator region of the lac operon. • In the presence of lactose, the repressor binds to it, and the repressor-lactose complexes float freely in the cytoplasm away from the DNA. In this situation, RNA polymerase can bind to the promoter, and the gene is transcribed. It makes beta-galactosidase which digests the lactose. • In the absence of lactose, the repressor binds to the operator DNA. The repressor is a large molecule, and when it is bound to the operator, RNA polymerase is blocked from reaching the promoter. The lac operon is not transcribed, and no beta-galactosidase is made. • If lactose appears, the operon is said to be “induced”. The lactose binds to the repressor, which then falls off the operator and allows transcription to occur.

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Genetic Analysis with Mutants • Jacob and Monod developed their model of lac regulation through the use of mutants. Later, biochemical techniques showed that their model was correct. • They isolated two main types of mutant: lac- mutants, which can’t use lactose as a food source, and “constitutive” mutants, in which the lac operon is always on regardless of external conditions. • They then tested these mutants alone and in combination with each other, with extensive use of merodiploids (partial diploids) to test for dominance. In particular, they created and used several F’ strains.

Constitutive Mutants • There are two ways of making a mutant strain where the lac operon is always on, regardless of whether lactose is present or not. • One way is an i- mutation: the lac. I gene does not produce a functional repressor protein. Since there is no repressor to bind to the operator, RNA polymerase is never inhibited, and the lac operon is always transcribed. • i- mutants are recessive: an i+ / i- heterozygote has normal gene regulation, because the wild type allele produces a normal repressor.

Operator Mutants • The operator is a region of DNA upstream from the structural genes that binds the promoter protein. It doesn’t make a protein product. • Mutations in the operator can only affect the gene to which it is attached. Such mutants are said to act in cis, or to be “cis-dominant”. • In contrast, repressor mutants make a protein which can move freely through the cell to any copy of the gene. Repressor mutants are “trans-acting”. (This terminology comes from cis and trans in organic chemistry. ) • Many operator mutants are constitutive, oc. The operator is mutated so that the repressor can no longer bind to it. Transcription occurs and the lac operon is on when no repressor is bound to the operator. • Demonstrating cis-dominance: oc z+ / o+ z- is always on: the normal lac. Z allele is attached to the constitutive operator. In contrast, oc z- / o+ z+ has normal regulation. The constitutive operator is attached to a defective lac. Z gene, so expression of this gene is not detected, while the normal operator is attached to a normal lac. Z gene, giving normal gene regulation.

Lac- Mutants • Most mutations in the 3 structural genes of the lac operon, lac. Z, lac. Y, and lac. A, affect only that particular gene. Thus, a lac. Z- mutant is usually lac. Y+ and lac. A+. • However, mutations in the control regions usually affect all 3 genes simultaneously. • One control mutation that affects all 3 genes is the super-repressed mutation, i. S. The repressor protein made by this mutant binds very tightly to the operator and does not bind to lactose. The effect is that the lac operon is always repressed, even when lactose is present. • i. S mutants are dominant: an i. S / i+ merodiploid shows the superrepressed phenotype: it is always off. A mixture of normal repressor proteins and super-repressor proteins will end up with the super-repressor sticking permanently to all copies of the operator that are present in the cell. In contrast, i- mutants are recessive.

Cis-Acting o. S Mutants • The operator can also be mutated to a super-repressed state, in which the operator binds so tightly to the repressor that it never gets released. • o. S mutants act in cis. For example, a o. S z+ / o+ z- strain is always off, because the only functional lac. Z gene is attached to the super-repressed operator. A o. S z- / o+ z+ strain shows normal regulation.

Summary of Lac Control Mutants • lac. I = repressor gene, makes repressor protein. Mutants act in trans. – i+ = normal regulation: ON in presence of lactose, OFF in absence of lactose. – i- = no repressor made, gene always ON, recessive. – is = super-repressor permanently bound to operator. Gene always OFF, dominant. • lac. O = operator region, controls expression of the attached lac. Z, Y and A genes. Mutants act in cis only. – o+ = normal expression of attached structural genes. – oc = constitutive, represor unable to bind to operator. Lac genes in cis are always ON. – os = super-repressed operator. Normal repressor protein stays permanently bound. The attached lac genes are always OFF.

A Few Examples • What is the lac. Z (beta-galactosidase) phenotype of these genotypes: normal expression, always on, or always off? • • • i+ o c z + i+ o c z is o c z + i+ o s z + / i + o c z i- o + z + / i - o c z i- os z- / is oc z-

Negative and Positive Regulation • As described above, the lac operon is negatively regulated: the regulatory protein (repressor) causes transcription to stop. • Positive regulation, where the regulatory protein causes transcription to start, is more common. • The lac operon also contains an example of positive regulation, called “catabolite repression”. E. coli would prefer to use glucose as its food source. In the presence of glucose, the lac operon (and other similar genes) are turned off, even if lactose is present in the medium.

Catabolite Repression • Catabolite repression uses a regulatory protein called CAP (catabolite activator protein). It also uses the small molecule cyclic AMP (c. AMP). • c. AMP is made from ATP. When the glucose level in the cell is high, the c. AMP level is low, because glucose inhibits synthesis of c. AMP. When the glucose level is low, the c. AMP level is high. • c. AMP combines with the CAP protein to form a complex that binds to part of the lac operon promoter. This complex bends the DNA in a way that makes it much easier for RNA polymerase to bind to the promoter. This allows transcription to occur, but only if the lac repressor isn’t present. • Thus, low glucose levels cause high c. AMP levels. When c. AMP is high, it combines with CAP The CAP-c. AMP complex then binds to the promoter to allow transcription to occur. • This is positive regulation because the binding of CAP to the DNA causes transcription to occur.

Eukaryotic Gene Regulation