Essay on Regulation of Gene Expression:- 1. Introduction to Regulation of Gene Expression 2. The Lac Operon 3. Bacteriophage Lambda (λ) 4. Gene Amplification during Development of Metazoans 5. Immunoglobulin Gene Rearrangement 6. Regulation of Messenger RNA Stability Provides another Control Mechanism.

Contents:

  1. Essay on Introduction to Regulation of Gene Expression
  2. Essay on the Lac Operon
  3. Essay on the Bacteriophage Lambda (λ)
  4. Essay on the Gene Amplification during Development of Metazoans
  5. Essay on the Immunoglobulin Gene Rearrangement
  6. Essay on the Regulation of Messenger RNA Stability Provides another Control Mechanism


1. Introduction to Regulation of Gene Expression:

A single ribosome is capable of translating about 400 codons in 10 seconds into a protein with a molecular weight of 40,000. Mammalian cells possess only about 1,000 times more genetic information than does the bac­terium E. Coli. Much of this additional genetic in­formation is probably involved in the regulation of gene expression.

There are only two types of gene regulation: positive regulation and negative regulation. When the expression of genetic information is quantita­tively increased by the presence of a specific regu­latory element, regulation is said to be positive’, whereas when the expression of genetic informa­tion is diminished by the presence of a specific regulatory element, regulation is said to be nega­tive.

The element or molecule mediating the nega­tive regulation is said to be a negative regulator, that mediating positive regulation is a positive regu­lator. A double negative has the effect of acting as a positive. Thus, an effector that inhibits the func­tion of a negative regulator will appear to bring about a positive regulation.

In many regulated sys­tems that appear to be induced, they are depressed at the molecular level. In biologic organisms, there are three types of temporal responses to a regulatory signal. These three responses are shown in the Fig. 26.5 as rate of gene expression in temporal response to an induc­ing signal.

A Type A response is characterized by an in­creased rate of gene expression that is dependent upon the continued presence of the inducing sig­nal. When the inducing signal is removed, the rate of gene expression is diminished to its basal level, but the rate repeatedly increases in response to the reappearance of the specific signal.

This type of response is commonly observed in many higher organisms after exposures to inducers such as ster­oid hormones.

A Type B response exhibits an increased rate of gene expression that is transient even in the con­tinued presence of the regulatory signal. After the regulatory signal has terminated and the cell has been allowed to recover, a second transient response to a subsequent regulatory signal may be observed.

This type of response may commonly occur during development of an organism when only the tran­sient appearance of a specific gene product is re­quired although the signal persists.

The Type (response exhibits an increased rate of gene expression that persists indefinitely even after the termination of the signal. The signal acts as a trigger in this pattern. Once the gene expres­sion is initiated in the cell, it cannot be terminated even in the daughter cells; it is, therefore, an irre­versible and inherited alteration.

The gene ox cistron is the unit of genetic infor­mation. It is the smallest segment of the DNA mol­ecule containing about 600 base pairs. The genetic message is carried in the sequence of bases along the DNA strand. These are arranged in orderly man­ner along the length of the DNA molecule in the chromosomes.

When a pair carries genes with the same characteristics, say tallness, then the individual is said to be homozygous with respect to that char­acter. When one of the pair tallness and the other gene shortness, the individual is heterozygous.


2. The Lac Operon:

Lac operon is nothing but structural gene + opera­tor gene.

A lactose analog which is capable of inducing the lac operon while not itself serving as a substrate for β-galactosidase is called a gratuitous inducer. The expression of some gene is constitutive i.e., they are expressed at a reasonably high rate in the absence of any specific regulatory signal.

The regulator gene controls the activity of the operator gene. The regulator gene induces the syn­thesis of protein macromolecules (probably RNA protein) called repressors.

The operon becomes active because the repressor system is itself inactivated. The phenom­enon is said to be De-repression. The operator locus is a region of double- stranded DNA of 27 base pairs long with a 2-fold rotational symmetry in a region that is 21 base pairs long.

The minimum effective size of an operator for lac repressor binding is 17 base pairs. The bind­ing occurs mostly in the major groove without in­terrupting the base-paired double helical nature of the operator DNA.

The binding of the inductor to the repressor molecule involves the amino acid residues in positions 74 and 75. The operator locus lies between the Promoter site, at which the DMA- dependent RNA polymerase attaches to commence transcription, and the beginning of the Z gene.

When attached to the operator locus the repressor molecule prevents the transcription of the operator locus and of the distal structural genes, Z, Y, A. Thus, the repressor molecule is a negative regula­tor and in its presence the expression of the Z, Y, and A genes is prevented. Normally 20-40 repressor molecules are present and one or two operator loci per cell are also found to be present.

The translation of the polycistronic mRNA can occur even before the transcription is completed. The depression of the lac operon allows the cell to synthesize the enzymes necessary to catabolize lactose as an energy source. Only for the RNA polymer­ase to attach at the promoter site, there must be the presence of the catabolite gene activation protein (CAP) to which cAMP is attached.

The bacterium accumulates cAMP only when it is starved for a source of carbon. In the presence of glucose or glyc­erol in concentrations sufficient for growth, the bac­teria will lack sufficient cAMP to bind to CAP.

Therefore, in the presence of glucose on glycerol, cAMP-saturated CAP is lacking, so that the DNA- dependent RNA polymerase cannot begin the tran­scription of the lac operon. In the presence of the CAP- cAMP complex on the promoter site tran­scription then takes place. Therefore, the CAP- cAMP regulator is acting as a positive regulator.

When the i gene is mutated so that its product, the lac repressor, is not capable of binding to op­erator DNA, the organism will exhibit constitutive expression of the lac operon.

An organism with an i gene mutation that prevents the binding of an inducer to the repressor will remain repressed even in the presence of the inducer molecule, because the inducer cannot bind to the repressor on the op­erator locus in order to depress the operon.


3. Bacteriophage Lambda (λ):

When a lamda infects a sensitive E. Coli, it injects its 45,000 base-pair, double stranded, linear DNA molecule into the cell (Fig. 26.8).

Depending upon the nutritional state of the cell, the lambda DNA will either integrate into the host genome (Lysogenic Pathway) and remain dormant until acti­vated, or it will commence replicating until it has made about 100 copies of complete, protein-pack-aged virus at which point it effects lysis of its host (Lytic Pathway).

The newly generated virus arti­cles can then infect other sensitive host.

When integrated into the host genome in its dormant state, lambda will remain in such a state until activated by exposure of its lysogenic bacte­rial host to DNA-damaging agents.

In response to such a noxious stimulus, the dormant bacteriophage becomes induced and begins to transcribe and sub­sequently translate those genes of its own genome which are necessary for its excision from the host chromosome, its DNA replication, and its protein cost and lysis enzymes. This event acts like a trig­ger of type C responses.

The switching event in lambda is centred around an eighty base pair region in its double- stranded DNA molecule referred to as the right op­erator (OR) Fig. 26.9A. The right operator is flanked on its left side by the structural gene for another regulatory protein called cro. When lambda is in its prophage state, the repressor gene is the only lambda gene that is expressed.

When the bacteri­ophage is undergoing lytic growth, the repressor gene is not expressed, but the cro gene, as well as many other genes in lambda, is expressed. That is,’ when the repressor gene is on, the cro gene is off, and when the cro gene is on, the repressor gene is off. These two genes regulate each other’s expres­sion.

The operator region can be subdivided into three discrete sites, each consists of 17 base pairs of similar but not identical DNA sequence (Fig. 26.9B). Each of these 3 sub-regions, (OR1, OR2, and OR3) can bind either repressor or cro proteins in the major groove of the DNA double helix.

The DNA region between the cro and repressor genes also contains two promoter sequences that direct the bind­ing of RNA polymerase in a specified orientation where it commences transcribing the adjacent genes.

One promoter directs RNA polymerase to begin transcription in the rightward direction and, thus, to transcribe cro and other distal genes, while the other promoter directs the transcription of the repressor gene in the leftward direction (Fig. 26.9B).

The product of the repressor gene, the 236 amino acid repressor protein, exists as a 2-domain molecule in which the amino-terminal domain binds to operator DNA and the carboxy-terminal domain promotes the association of one repressor protein with another to form a dimer.

A dimer of repressor molecules binds to operator DNA much more tightly than does the monomeric form (Fig. 26.10-A to C). The product of the cro gene, the 66-amino acid cro protein has a single domain but also binds the operator DNA more tightly as a dimer (Fig. 26.10D).

In a lysogenic bacterium i.e., a bacterium con­taining a lambda prophage, the lambda repressor dimer binds preferentially to OR1 but in doing so by a cooperative interaction, enhances the binding of another repressor dimer to OR2 (Fig 26.11). The affinity of repressor of OR1 is the least of the three operator sub-regions.

The binding of repressor to OR1 has two major effects. The occupation of ORI by repressor blocks the binding of RNA polymer­ase to the rightward Promoter and thereby prevents’ the expression of the cro gene. Secondly, repressor dimer bound to OR1 enhances the binding of repressor dimer OR2.

The binding of repressor to OR2 has the important effect of enhancing the bind­ing of RNA polymerase to the leftward promoter that overlaps OR2 and thereby enhances the tran­scription and subsequent expression of the repressor gene.

Thus, the lambda repressor is both a negative regulator by preventing transcription of the cro gene, and a positive regulator, by enhancing the transcription of its own gene, the repressor gene. This dual effect of repressor is responsible for the stable state of the dormant lambda bacterio­phage.

When a DNA damaging signal, such as ultra­violet light, strikes the lysogenic host bacterium, fragments of signal-stranded DNA are generated that activate a specific protease coded by a bacte­rial gene and referred to as rec A. The activated rec A protease hydrolyzes the portion of the repressor protein that connects the amino-terminal and carboxytenninal domains of that molecule.

Such cleavage of the repressor domains causes the repressor dimers to dissociate, which in turn causes a dissociation of the repressor molecules from OR2 and eventually from OR1. The effects of removal of repressor from OR1 and OR2 are speculated.

RNA polymerase immediately has access to the right- ward promoter and begins transcribing the cro gene, and the enhancement effect of the repressor at OR2 on leftward transcription is lost.

The cro protein translated from the newly tran­scribed cro gene also binds to the operator region as dimers, but its order of preference is the opposite of that of repressor. That is, cro binds most lightly to OR3, but there is no cooperative effect of cro at OR3 on the binding of Cro to OR2. At increasingly higher concentrations of Cro, the protein will bind to OR2 and eventually to OR1.

The occupancy of OR3 Cro immediately turns off the transcription from the leftward promoter and hence, prevents any further expression of the repressor gene. Therefore, the switch is completely effected. The Cro gene is now expressed and the repressor gene is fully turned off.

When Cro repressor concentration becomes very high, it will eventually occupy OR’ and in doing so turn down the expression of its own gene, a process that is nec­essary to effect the final stages of the lytic cycle.


4. Gene Amplification during Development of Metazoans:

During early development of metazoans, there is an abrupt increase in the need for specific genetic molecules such as ribosomal RNAs and messenger RNA molecules for proteins that make up such or­gans as the egg-shell. One way to increase the rate at which such molecules can be formed is to in­crease the number of genes available for transcrip­tion of these specific molecules.

Among the repeti­tive DNA sequences are thousands of copies of ri­bosomal RNA genes and tRNA genes. These genes preexist repetitively in the genomic material of the gametes and, thus, are transmitted in high copy number from generation to generation.

In some spe­cific organisms such as the fruit fly (Drosophila), there occurs during oogenesis an amplification of a few pre-existing genes, such as these for the cho­rion (eggshell) proteins, S36 and S38. Subsequently, these amplified genes, presumably generated by a process of repeated initiations during DNA synthe­sis provide multiple sites for gene transcription.

In recent years, it has been possible to pro­mote the amplification of specific genetic regions in cultured mammalian cells. In some cases, a sev­eral thousand-fold increase in the copy number of specific genes can be achieved over a period of time involving increasing doses of selective drugs.

In fact, it has been demonstrated in patients receiv­ing methotrexate for treatment of cancer that ma­lignant cells can develop drug resistance by in­creasing the number of genes for dihydrofolate re­ductase, the target of methotrexate.


5. Immunoglobulin Gene Rearrangement:

a. The coding segments responsible for the generation of specific protein molecules are frequently not contiguous in the mam­malian genome. Immunoglobulin mole­cules consist of two types of polypeptide chains, light (L) and heavy (H) chains.

The L and H chains are each divided into N-terminal variable (V) and carboxy-terminal constant (C) regions. The V regions are responsible for the recognition of an­tigens (foreign molecules) and the con­stant regions for effector functions that de­termine how the antibody molecule will dispense with the antigen.

b. There are three unlinked families of genes responsible for immunoglobulin molecule structure. Two families are responsible for the chains (A, and k chains) and one family for heavy chains.

c. Each light chain is encoded by three dis­tinct segments, the variable (VL), the join­ing (JL), and the constant (CL) elements. The mammalian haploid genome contains over 500 VL segments, five or six JL seg­ments, and 10 or 20 CL segments.

d. A VL segment is brought from a distant site on the same chromosome to a posi­tion closer to the region of the genome containing the JL and CL segments dur­ing the differentiation of a lymphoid B cell. This DNA 4 rearrangement then allows the VL, JL and CL segments to be tran­scribed as a single mRNA precursor and subsequently processed to generate the mRNA for a specific antibody light chain.

The immunity system can generate a di­verse library of antigen specific immu­noglobulin molecules by rearrangement of the various VL, JL, and CL segments in the genome. The DNA rearrangement is referred to as V-J joining of the light chain.

e. The heavy chain is encoded by four gene segments the VH, the D (Diversity), the JH, and the CH DNA segment. The vari­able region of the heavy chain is gener­ated by joining the VH with a D and a JH segment. The resulting VH-D-JH-DNA re­gion is in turn linked to a CH gene. These CH genes (Cµ, Cδ, Cγ3 Cy1, Cy2b, Cy, Cα and Ce) determine the immunoglobu­lin class or subclass-lgM, lgG, IgA, etc. – of the immunoglobulin molecule.

f. A B cell that secretes antibody to a specific antigen will secrete antibodies of dif­ferent classes having the same antigen specificity but different biologic roles during the differentiation. The different classes of immunoglobulin’s contain the same light chains and VH regions but dif­ferent CH regions.

Thus, a single B cell and its derivatives can undergo class switching which is the result of second type of DNA rearrangement occurring dur­ing differentiation of the immunity sys­tem. The V-J joining for light chain ex­pression and the V-D-J joining for heavy chain expression precede the class switching DNA rearrangement.

V-J Joining:

a. In the undifferentiated cell (e.g., germ line cell), the K-J gene (Jk) is closely linked to the Ck gene, but the gene segment for the k-variable region (Vk) is quite distant on the same chromosome.

b. Both the Vk-Jk and the similar Vλ-Jλ gene rearrangements to involve two short con­served sequence that exist in the direc­tion 3′ to the V-segment and 5′ to the J- segment, close to the point of recombina­tion.

c. There are two striking features of these conserved sequences. First, the conserved sequences of the JL segments are inverse complements of the conserved sequences in the VI. segments. Second, the length of the non-conserved sequence separating the heptamers and the monomers is highly conserved.

d. The variable region of the heavy chain involves three DNA segments, VH, D, and JH, which must be joined in a process in­volving two DNA rearrangements since all three segments are separated.

Class Switching:

1. Since differentiation proceeds and immu­noglobulin production switches from IgM to IgA the V-D-J region of the parent B cell must be rearranged with C0 gene to permit the transcription of an mRNA pre­cursor for an a-chain containing the same antigen-specific variable region.

2. The temporal order of the class switching’ is unidirectional within the physical or­der from left to right. Rearrangement of the CH genes seems to involve deletion of those CH genes 5′ to the CH gene joined to the V-D-J region.

The switch sites are different for different class switches but involve conserved sequences occur­ring in the appropriate regions of the genes to be rearranged.

Transcription Control:

a. Glucocorticoids regulate gene expression. Once they enter the mammalian cell, they bind to a steroid specific receptor molecule that undergoes a conformational change in the cytoplasm and enters the nucleus. The glucocorticoid receptor complex in the nucleus binds to a specific receptor-recognition site on DNA, a few hundred base pairs 5′ upstream from the transcrip­tion start site, for steroid-responsive genes.

The receptor recognition site on DNA in­fluences the efficiency of utilization of the promoter by RNA polymerase and thereby influences the expression of the steroid responsive gene.

b. When an organism or its cultured cells are exposed to metal ions, such as zinc or cad­mium, there is an accelerated rate of tran­scription of the metallothionein gene and a subsequent increase in the metallo­thionein protein to bind the potentially toxic heavy metal.

Another structural gene, such as that for thymidine kinase, can be ligated to this ‘metallothionein promoter region’ and the synthetic con­struct introduced to cultured cells, a small number of which will integrate the DNA into its own genome. When those cells are exposed to heavy metals, the metallo­thionein promoter region effects an induc­tion of thymidine kinase.

c. There are many more primary transcripts in the nucleus than are represented as messenger RNA molecules in the cytoplasm. There must exist regulatory decisions as to which transcripts will ultimately be expressed and which will be discarded. There is no information available regard­ing the mechanism of this process.


6. Regulation of Messenger RNA Stability Provides another Control Mechanism:

a. Most mRN As in mammalian cells are very stable. Changes in the stability of a spe­cific mRNA have the major effects in bio­logic processes.

b. Messenger RNAs exist in the cytoplasm as ribonucleoprotein particles (RNPs). Some of these proteins protect the mRNA from digestion by nucleases, while others under certain conditions, promote nucle­ase attack. Certain effectors, such as hor­mones, may regulate mRNA stability by increasing or decreasing the amount of the proteins.

c. Then ends of mRNA molecules are in­volved in mRNA stability. The 5′ cap structure in eukaryotic mRNA prevents attack by 5′ exonucleases, and the poly (A) tail prohibits the action of 3′ exonucleases. In mRNA molecules, a sin­gle endonucleolytic cut allows exonucleases to attack and digest the en­tire molecule.

Other structures in the 5′ noncoding sequence (5′ NCS) the coding region, and the 3’NCS are thought to pro­mote or prevent this initial endonucleo­lytic action.

d. Structures at the 3′ end including the poly (A) tail enhance or diminish the stability of specific mRNAs. The absence of poly (A) tail is associated with rapid degrada­tion of mRNA and the removal of poly (A) from some RNAs results in their destabilization.

e. In AU-rich regions, many of which con­tain the sequence AUUUA which appears in mRNAs that have a very short half-life including some encoding oncogene pro­teins and cytokines.


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