In this article we will discuss about:- 1. The Inducible and Repressible Systems 2. Transcriptional and Translational Control 3. Regulation of E.coli Tryptophan Operon 4. Regulation of Transcription in Eukaryotes 5. Promoters and Enhancers 6. Transcriptional Activators 7. Regulation by Alternative Splicing of RNA Transcript 8. Regulation at the Level of Translation 9. Epigenetic Control of Genetic Regulation.
The Inducible and Repressible Systems:
In the inducible system when inducer is absent, the repressor binds to the operator and blocks it; RNA polymerase cannot move along DNA so that very small amounts of mRNA if at all, are synthesised by the structural genes.
But when lactose is present in the medium as an inducer, the operon is induced to synthesise large quantities of the enzymes required in the transport and catabolism of lactose. In this case the operon functions because the repressor gets bound to the inducer molecule, the operator becomes free, and structural genes synthesise mRNA.
The lac operon provides one example of an inducible system in which the existence of the nutrient in the medium induces synthesis of large amounts of enzymes needed for the catabolism of that nutrient. Such systems therefore, operate in the degradation of exogenous substrates in catabolic processes.
In repressible systems the operon controls synthesis of proteins or enzymes needed for anabolic reactions. Such an operon continues to function normally until there is an excess of the products.
When the end product is in excess it functions as a repressing metabolite called co-repressor. In this system the repressor is inactive by itself. But when the co-repressor binds to the repressor to form a repressor-co-repressor complex which attaches to the operator, the structural genes cannot transcribe mRNA (Fig. 16.3).
In a repressible system the operon is negatively controlled. In addition to the histidine operon already described, the tryptophan operon in E. coli also functions as a repressible operon. When there are normal concentrations of tryptophan in a cell, the operon is functional or de-repressed. But when tryptophan is in excess, it acts as a co-repressor and binds to the inactive repressor.
The complex attaches to the operator and prevents mRNA synthesis by structural genes. As the concentration of tryptophan decreases, it causes the repressor molecules to remain free, the operator becomes unbound, and the structural genes transcribe mRNA. Thus the operon again becomes de-repressed.
Transcriptional and Translational Control:
The lactose operon demonstrates that the control of transcription involves interaction of regulatory proteins with specific DNA sequences, and this is broadly applicable to eukaryotes as well. Regulatory sequences such as the operator are called cis-acting control elements because they bring about expression of only linked genes on the same DNA molecule.
In contrast, repressor proteins are called trans-acting factors because they can influence the expression of genes located on other chromosomes within the cell. Furthermore, the lac operon is considered as an example of negative control of gene expression because binding of the repressor inhibits transcription. There are however, other examples where trans-acting factors are activators (positive control) of transcription.
Positive control of transcription has been demonstrated in E. coli through studies on the effect of glucose on the expression of genes encoding enzymes that lead to breakdown (catabolism) of other sugars, such as lactose. Lactose provides an alternative source of carbon and energy.
As long as glucose is available, it is preferentially utilised, with the result that enzymes involved in catabolism of alternative energy sources are not expressed. That means, if E. coli are grown on a medium that provides both glucose and lactose, the lac operon is not induced, and only glucose is used by cells of E.coli. Thus, glucose represses the lac operon even in the presence of the normal inducer, lactose.
Repression by glucose, also called catabolite repression is actually mediated by a positive control system which is determined by levels of cyclic AMP. (cAMP) (Fig. 16.4). In bacteria cAMP is produced from ATP by enzyme adenylyl cyclase. The conversion of ATP to cAMP is regulated in such a way that levels of cAMP increase when glucose levels drop. Cyclic AMP then binds to a transcriptional regulatory protein called catabolite activator protein (CAP).
The binding of cAMP to CAP stimulates the binding of CAP to its specific DNA sequences. In the lac operon this specific DNA sequence is located approximately 60 bases upstream of the transcription start site. CAP then interacts with the alpha subunit of RNA polymerase, and that facilitates the binding of polymerase to the promoter and activating transcription.
Regulation of E.coli Tryptophan Operon:
Genes of the amino acid tryptophan (trp genes) are considered as repressible genes in which the presence of the metabolite (trp) in the environment turns off the expression of its structural genes. Tryptophan acts as a co-repressor. Regulation of the trp operon occurs in two ways.
In the first, the expression of the five structural genes E, D, C, B, and A that code for enzymes involved in the synthesis of tryptophan, is controlled by a specific regulatory gene. The regulatory gene codes for a specific protein called repressor. The repressor by itself is inactive, but when it becomes complexed with tryptophan (co-repressor) it becomes activated.
The activated repressor-co-repressor complex hinds to a specific region of DNA, the operator situated adjacent to the structural genes that are being regulated. This blocks the movement of RNA polymerase towards structural genes.
The structural genes, operator and promoter regions together constitute the operon. Thus, when tryptophan is present in the environment, the repressor forms a complex with tryptophan, then binds with operator and prevents transcription of the structural genes.
On the contrary, when tryptophan is lacking in the environment, the repressor remains free and inactive, does not bind with operator, resulting in transcription of structural genes and synthesis of tryptophan. This is referred to as a negative control system because the repressor which is product of the regulatory gene acts to turn off transcription of structural genes (Fig. 16.5).
The second mechanism called attenuation, regulates the expression of tryptophan structural genes by controlling the ability of RNA polymerase to continue elongation over a specific nucleotide sequence. This mechanism operates when high levels of tryptophan are available. There is attenuation of regulation by a sequence that terminates transcription prematurely.
This sequence or region of attenuation is located 162 nucleotides downstream of the transcription start site, that is the first structural gene. Transcription terminates in this region if tryptophan is available, before RNA polymerase reaches the first structural gene. In other words, attenuation occurs if the specific amino-acylated tRNA is available. If not, transcription continues, producing a functional trp mRNA.
Transcription is initiated at the promoter region producing what is referred to as the leader transcript. The leader RNA contains a start and a stop signal for protein synthesis. Since prokaryotes lack a nuclear membrane, transcription and translation can occur simultaneously, unlike eukaryotes where there is spatial separation of transcription (in nucleus) and translation (in cytoplasm).
Therefore, while the leader RNA is being synthesised, ribosomes begin translation at the 5′ end. This results in a short peptide chain while the RNA polymerase is transcribing the leader region. If tryptophan-tRNA is available, synthesis of the peptide chain will continue, until the ribosome reaches the stop signal present in the leader RNA.
However, if there is not sufficient tryptoph an -tRNA, the leader RNA will not be translated into peptide, and the ribosome will be arrested at the tryptophan codons in the leader RNA, without reaching the stop signal.
Besides the stop and start sequences, the leader RNA contains 4 regions which have complementary sequences which enable formation of stem and loop structures by base pairing.
Region 1 can form base pairs with region 2; region 2 can form base pairs on both sides either with region 1 or with region 3; region 3 can likewise form base pairs with region 2 or with region 4; region 4 can base pair only with region 3. Therefore, 3 possible stem/loop structures can form in the RNA transcript (Fig. 16.6).
When region 3 base pairs with region 4, it generates a signal for attenuation, that is, premature termination of transcription. However, it must be noted that if stem/loop has already been formed in the region preceding region 3, then region 3 will not be available to base pair with region 4.
Another important point to note is that, if the ribosome is translating in region 2, then region 2 would not be available for base pairing with region 1 or with region 3. In that situation region 3 will be free to base pair with region 4.
Base pairing only between regions 3 and 4 to form stem/loop signals RNA polymerase to terminate transcription. This implies that when sufficient amount of tryptophan-tRNA is available to translate the leader RNA, it will stop transcription (attenuation) prematurely, and structural genes will not be transcribed, In contrast, if tryptophan-tRNA is lacking or insufficient to translate the leader RNA, there will be no attenuation.
In that case the ribosome will stop at the two trp codons in region 1, thus leaving region 2 available to base pair with region 3. That means region 3 would not be available to base pair with region 4, which is an essential requirement for signaling RNA polymerase to terminate transcription. In absence of attenuation then structural genes will be transcribed (Fig. 16.7).
Regulation of Transcription in Eukaryotes:
The control of expression of eukaryotic genes is more complex than in prokaryotes, and is primarily at the level of initiation of transcription. In general, transcription in eukaryotic cells is controlled by proteins that bind to specific regulatory sequences and modulate the activity of RNA polymerase.
In the many different cell types of multicellular eukaryotic organisms, regulation of gene expression is accomplished by the combined actions of multiple different transcriptional regulatory proteins, by methylation of DNA, and packaging of DNA into chromatin.
Promoters and Enhancers:
In bacteria, transcription is regulated by the binding of proteins to cis-acting sequences, as in the lac operon, that control transcription of adjacent genes (z, y, a). Similar cis-acting sequences regulate the expression of eukaryotic genes. The method of identifying these sequences is based on the use of gene transfer assays by which the activity of supposed regulatory regions of cloned genes are studied (Fig. 16.8).
The regulatory sequence is ligated to a reporter gene that encodes an easily detectable enzyme. The reporter gene is transferred into cultured cells (transfection). The expression of the reporter gene indicates biological activity of the regulatory sequence and provides a sensitive assay for the ability of the cloned regulatory sequences to direct transcription.
The two core promoter elements, the TATA box and the Inr sequence in genes transcribed by RNA polymerase II serve as specific binding sites for transcription factors. Inr is the initiator sequence spanning the transcription start site in promoters of many genes transcribed by RNA polymerase II.
Other cis-acting sequences function as binding sites for a variety of regulatory factors that control expression of individual genes. These cis-acting regulatory sequences are usually located upstream of the TATA box. Interestingly, two regulatory sequences commonly found in eukaryotic gene were found to be present in the promoter of the herpes simplex virus gene that encodes thymidine kinase.
These two sequences are located about 100 base pairs upstream of the TATA box, and their consensus sequences are CCAAT and GGGCGG (called the GC box). The binding of specific proteins to these sequences has been shown to initiate transcription.
In contrast to the CCAAT and GC boxes in the thymidine kinase promoter of herpes simplex virus, the regulatory sequences of several mammalian genes are located further away, upto 10 kilo-bases, from the transcription start site. These sequences are called enhancers and were first described in the virus SV40. Like promoters, enhancers function by binding transcription factors that act by regulating RNA polymerase.
Transcription factors bound to distant enhancers function by the same mechanisms as those bound adjacent to promoters, that is, the cis-acting regulatory sequences. The binding of specific transcriptional regulatory proteins to enhancers is responsible for the control of gene expression during development, differentiation and in response of cells to hormones and growth factors.
Transcriptional Activators:
Among the most thoroughly studied transcription factors are the transcription activators, which bind to regulatory DNA sequences and stimulate transcription. Transcriptional activators consist of two domains, one region binding to DNA to anchor the factor to the proper site on DNA; the other activates transcription by interacting with other components of the transcriptional system.
Detailed studies have revealed that the DNA-binding domains of many of these proteins are related to one another. The zinc finger domains contain repeats of cysteine and histidine residues that bind zinc ions and fold into finger-like loops that bind DNA. Transcription factors of the steroid hormone receptors contain zinc finger domains.
The steroid hormone receptors regulate gene transcription in response to hormones estrogen and testosterone. The activation domains of transcription factors are not as well characterised as their DNA binding domains.
Regulation by Alternative Splicing of RNA Transcript:
The primary transcript of some genes could be spliced in alternative ways to yield different products. Even when the same promoter is used to transcribe a gene, different cell types can produce different quantities of a protein, or even a different protein. This could result from differences in the mRNA produced or from processing of mRNA. This can be achieved when the same transcript from one cell type is spliced differently from the transcript in another type of cell.
The protein- coding exons may be the same in the different cell types, but the splicing pattern of the transcript may be different. In that case the protein is identical, but the rate of synthesis is different, because the mRNA molecules are not translated with the same efficiency.
In other cases, the protein-coding part of the transcript has a different splicing pattern in each cell type, with the result that the mRNA molecules produced code for proteins that are not identical even though they share certain exons.
Transcripts of the human genome are frequently spliced in alternative ways. Owing to this, the approximately 30,000 human genes may encode 64,000 to 96,000 different proteins. Alternative RNA processing is considered to be one of the principle sources of human genetic complexity. For example, the human insulin receptor gene undergoes alternative splicing that results in the inclusion or exclusion of exon number11 in the mRNA (Fig. 16.9).
The resulting forms of the polypeptide chain differ in length by 12 amino acids. In liver cells, all the 20 exons are present in mRNA for the long form of the receptor protein (Fig. 16.9, Part A), whereas in skeletal muscle exon 11 is eliminated along with the flanking introns and excluded from the mRNA for the short form (Fig. 16.9, Part B).
The long form of the receptor shows low affinity for insulin and is expressed in tissues such as the liver that are exposed to relatively higher concentrations of insulin. The short form of the protein has a high affinity for insulin and is expressed preferentially in tissues such as the skeletal muscle that are normally exposed to lower levels of insulin.
Thus, alternative splicing provides a mechanism for generating proteins with different properties from the same gene. The Dscam gene of Drosophila could give rise to approximately 38016 different proteins by alternative splicing. The actual number of proteins synthesised is not known. The human genome that contains 30,000 to 40,000 genes produces different proteins whose number is several times greater, by alternative splicing.
Unlike genes of Drosophila and lower worms, human genes are distributed over a large region of the genome, and the primary mRNA transcripts are very long. Alternative splicing of most human genes leads to multiple protein products. About one third of all human genes are believed to undergo alternative splicing.
Among genes that are alternatively spliced, the average number of distinct mRNAs produced from the primary transcript ranges between 2 and 7. The average number of different mRNAs per gene across the genome is in the range of 2 to 3, which includes genes that produce a single mRNA as well as those that produce multiple different mRNAs. Thus alternative splicing greatly increases the number of protein products that can be encoded from a relatively small number of genes.
Regulation at the Level of Translation:
In eukaryotic cells, transcription and translation are uncoupled in the process of gene expression. This permits regulation at the level of translation independently from transcription.
The major types of translational control are: inability of a mRNA molecule to be translated under certain conditions; regulation of the overall rate of protein synthesis; inhibition or activation of translation by small regulatory RNAs that undergo base pairing with the mRNA; activation of previously un-translated cytoplasmic mRNAs.
In the case of translational control by small regulatory RNAs, usually the regulatory RNAs are complementary in sequence to part of the mRNA whose translation they control. An RNA sequence that is complementary to a mRNA is called an antisense RNA. The antisense regulating RNAs act by pairing with the mRNA. It can either activate or inhibit translation. Small regulatory RNAs can also regulate translation.
Epigenetic Control of Genetic Regulation:
Epigenetic phenomena are alternative states of gene activity that are heritable, but do not follow Mendelian rules of inheritance, are not explained by mutation, changes in gene sequence or normal developmental regulation. They are changes brought about in gene expression by heritable chemical modifications in DNA.
The prefix epi means “besides” or “in addition to”. Therefore, epigenetic refers to heritable changes in gene expression that are not associated with changes in the DNA sequence, but with something “in addition to” the DNA sequence, usually either chemical modification of the DNA bases or proteins bound with DNA.
It is now clear that many epigenetic phenomena occur largely via changes in chromatin structure. In general, methylation of DNA is associated with turning off gene expression. However, some organisms that clearly exhibit epigenetic effects, for example Drosophila, do not have DNA methylation. Modifications of histones and non-histone chromosomal proteins have also been implicated in epigenetics.