Regulation of Gene Expression

Regulated expression of genes is required for development, differentiation and adaptation.

Gene expression is:
1 · regulated during ontogeny and differentiation of the organism and its cellular components
2 · needed so the organism can adapt to its environment and conserve energy and nutrients
3 · needed to respond to complex environmental challenges

Transcription level · gene amplification · gene rearrangement · post-transcriptional modifications · RNA stabilization.

The process works through the interaction of specific binding regulatory proteins with various regions of DNA, which have a positive or negative effect on transcription, and can result in tissue-specific gene expression. It is influenced by hormones, heavy metals and chemicals.

Type A response — expression rises while the inducer is present and falls when it is withdrawn; the lac operon is the classic example.
Type B response — expression rises and remains elevated even after the inducer is removed.
Type C response — occurs during the development of differentiated function in a tissue or organ, and is essentially irreversible.

Negative regulation: the regulatory element is present and expression is decreased. The protein responsible is a repressor.

Positive regulation: the regulatory element is present and expression is increased. That mediating positive regulation is a positive regulator or ACTIVATOR.

Derepression is the removal of a repressor — and note the logic that follows: a double negative has the effect of acting as a positive.

Inducible genes have relatively low basal rates of transcription and are switched up when needed.

Housekeeping genes are expressed constantly, at a roughly constant level, because their products are always needed.

A constitutive mutation is one that causes a normally inducible gene to be expressed all the time — for example, a mutation that destroys the repressor or its binding site.

An operon can be regulated by a single promoter or regulatory region. It is a cluster of genes transcribed together as one unit.

A cistron is the genetic unit encoding one polypeptide — the “one cistron, one subunit” concept, which refines the older “one gene, one enzyme”.

Polycistronic mRNA is transcribed into one large mRNA molecule that contains multiple independent translation start (AUG) and stop (UAA) codons for each cistron. The result is coordinate expression — all the genes rise and fall together.

The lac repressor has four identical subunits of molecular weight 38,000 and a high affinity for the operator locus.

The operator locus is a region of double-stranded DNA 27 base pairs long, with a twofold rotational symmetry and an inverted palindrome. LacI repressor binding covers 17 base pairs, and only two subunits of the repressor appear to bind.

Addition of lactose or of a gratuitous inducer such as IPTG causes the inducer to bind the repressor, which then releases the operator: an inducer DEREPRESSES the lac operon.

The kinetics are fast: at high concentrations of lactose with no or very low glucose, lac-specific mRNAs are fully induced within 5–6 minutes, and β-galactosidase protein is maximal within 10 minutes. This is the type A response.

Translation of the polycistronic mRNA can occur even before transcription is completed — the prokaryotic coupling of Unit 24.

When exposed to both lactose and glucose as sources of carbon, bacteria first metabolize the glucose and then temporarily stop growing, until the genes of the lac operon become induced and they gain the ability to metabolize lactose as a usable energy source.

This is CATABOLITE REPRESSION, and it is mediated by a catabolite gene activator protein (CAP) in conjunction with cAMP. This protein is also referred to as the cAMP regulatory protein (CRP).

When glucose or glycerol is present in concentrations sufficient for growth, the bacteria will lack sufficient cAMP to bind to CAP, because the glucose inhibits adenylyl cyclase, the enzyme that converts ATP to cAMP.

Without cAMP, CAP cannot bind DNA and cannot activate transcription. The CAP-cAMP regulator acts as a POSITIVE regulator.

So the operon is controlled by two regulatory proteins: one that acts positively (the cAMP-CRP complex) and one that acts negatively (the LacI repressor). Maximal activity requires lactose present AND glucose absent.

The genetic switch of bacteriophage lambda chooses between the lysogenic pathway — the virus integrates and lies dormant as a prophage — and the lytic pathway — the virus replicates and destroys the host.

Exposure of its lysogenic bacterial host to DNA-damaging agents causes the dormant bacteriophage to become “induced”, switching from the prophage state to a lytic infection.

The right operator (O_R) lies in the DNA region between the cro and repressor genes, and is subdivided into three discrete, evenly spaced, 17-bp cis-active DNA elements whose nucleotide sequences are similar but not identical. Each may be bound by either cI or Cro proteins, and the relative affinities of cI and Cro for each of the sites varies.

There are two promoter sequences: one directing rightward transcription of cro and other distal genes, the other leftward transcription of the repressor gene.

The repressor protein (cI) is 236 amino acids, 27 kDa. The Cro protein is 66 amino acids, 9 kDa, a single domain mediating both operator binding and dimerization.

When the repressor gene is on, the cro gene is off — the prophage state; when the cro gene is on, the repressor gene is off — lytic growth.

In a lysogenic bacterium the repressor's affinities run O_R1 > O_R2 > O_R3. Binding of repressor to O_R2 both blocks cro transcription and activates its own gene.

Ultraviolet light strikes the lysogenic host bacterium → fragments of single-stranded DNA are generated → these activate a specific protease coded by a bacterial gene (recA) → the protease hydrolyzes the repressor protein → dissociation of the repressor molecules from O_R2 and O_R1 → RNA polymerase immediately has access to the rightward promoter.

Cro protein then binds the operator region as a dimer, expression of other lambda genes begins as part of the lytic cycle, and later Cro comes to occupy O_R1, reducing expression and effecting the final stages of the lytic cycle.

Chromatin remodeling is an important aspect of eukaryotic gene expression. Chromatin structure provides an additional level of control of gene transcription — one that prokaryotes, having no nucleosomes, do not possess.

Acetylation and deacetylation occur on lysine residues in the amino terminal tails of histone molecules.

Acetylation reduces the positive charge of these tails, which decreases the binding affinity of histone for the negatively charged DNA, causing disruption of nucleosomal structure. That gives access of transcription factors to cognate regulatory DNA elements and enhances binding of the basal transcription machinery to the promoter.

Histone deacetylation would have the opposite effect.

Methylation of deoxycytidine residues in the sequence 5′-mCpG-3′ silences genes.

In mouse liver, only the UNmethylated ribosomal genes can be expressed, and many animal viruses are not transcribed when their DNA is methylated.

Remodelling proceeds through multiple protein-binding DNA elements and the binding of transcription factors to these elements, which can disrupt the structure of the nucleosome, prevent its re-formation, or recruit further remodelling activities.

As Unit 24 put it: promoter accessibility, and hence PIC formation, is often modulated by nucleosomes, and nucleosome eviction by chromatin-active coregulators facilitates PIC formation and transcription.

Certain DNA elements enhance or repress transcription of eukaryotic genes. The classic example is the simian virus 40 (SV40) enhancer.

Enhancer binding proteins interact with a plethora of other transcription proteins, and unlike promoter elements, enhancers work at great distance — 1000 to 10⁵ bp — and in either orientation.

The β-interferon gene is induced upon viral infection of mammalian cells. Its enhancer element is located between nucleotides −110 and −45 and contains four distinct clustered cis elements, bound by four trans factors:

1 · NF-κB
2 · a member of the IRF (interferon regulatory factor) family
3 · the heterodimeric leucine zipper factor ATF-2/c-Jun
4 · HMG I(Y), a ubiquitous architectural transcription factor

HMG I(Y) induces a significant bend in the DNA, and along with the three trans factors, by inducing a series of critically spaced DNA bends, brings about the formation of a unique, stereospecific, three-dimensional structure in which all four factors are active — the ENHANCEOSOME.

Combinations of DNA elements and associated proteins provide diversity in responses: the same factor may activate one gene and, in different company, inhibit another — gene C is inactivated by the combination of factors 1, 5 and 3, because factor 5 precludes the essential binding of factor 2 to DNA.

Tissue-specific expression may result from the action of enhancers or repressors.

Reporter genes are used to define enhancers and other regulatory elements: a DNA fragment of 2 kb of 5′-flanking DNA and its cognate promoter is ligated to a reporter, transfected, and the reporter activity detected; addition of one or more hormones then reveals functional hormone response elements (HRE).

Transcription domains can be defined by locus control regions and insulators.

Several motifs mediate the binding of regulatory proteins to DNA: the helix-turn-helix, the zinc finger, and the leucine zipper.

1 · Binding must be of high affinity to the specific site and of low affinity to other DNA.
2 · Binding is usually by a dimer or higher multimer, to a site with matching symmetry.
3 · The protein-DNA interactions are maintained by hydrogen bonds and van der Waals forces.

Illustrated by the Cro protein and its binding to DNA.

The Cro monomer consists of three antiparallel β sheets (β1–β3) and three α-helices (α1–α3). The α3 and α2 helices are held at about 90 degrees to each other by a turn of four amino acids, and the α3 helix of Cro is the DNA recognition surface.

The distance between comparable points on the two DNA α-helices is 34 Å — exactly one turn of B-DNA, so a dimer places one recognition helix in each of two successive major grooves.

The zinc finger motif — a small domain folded around a zinc ion, which stabilises the structure so that a short α-helix can enter the major groove. Zinc fingers occur in tandem arrays, each finger reading a few base pairs.

The leucine zipper motif — a helical wheel consisting of seven spokes that correspond to the seven amino acids of each turn, in which leucine residues occur at every seventh position. The leucines line up along one face of the helix and interdigitate with those of a partner helix — hence “leucine zippers”. The zipper mediates dimerisation; an adjacent basic region does the DNA binding.