In this article we will discuss about the Transcriptional and Post-Transcriptional Regulation in Prokaryotes.
Transcriptional Regulation of Gene Expression in Prokaryotes:
Gene transcription is regulated in bacteria through a complex of genes termed operon. These are transcriptional units in which several genes, with related functions, are regulated together. Other genes also occur in operons which encode regulatory proteins that control gene expression. Operons are classified as inducible or repressible.
Inducible and Repressible System:
The β galactosidase in E. coli is responsible for hydrolysis of lactose into glucose and galactose.
If lactose is not supplied to E. coli cells, the presence of β galactosidase is hardly detectable. But as soon as lactose is added, the production of β galactosidase enzyme increases. The enzyme falls as quickly as the substrate (lactose) is removed.
Such enzymes whose synthesis can be induced by adding the substrate are known as inducible enzymes and the genetic system responsible for the synthesis of such an enzyme is called inducible system. The substrate whose addition induces the synthesis of an enzyme is inducer.
In some other cases, the situation is reverse. For instance, when no amino acids are supplied from outside, the E. coli cells can synthesize all the enzymes needed for the synthesis of different amino acids. However, if a particular amino acid, for instance, histidine, is added, the production of histidine synthesizing enzyme falls.
In such a system, the addition of the end product of biosynthesis checks the synthesis of the enzymes needed for the biosynthesis. Such enzymes whose synthesis can be checked by the addition of the end product are repressible enzymes and the genetic system is known as repressible system. The end product, the addition of which check the synthesis of the enzyme is co-repressor.
A class of molecules called repressors are found in cells and these repressors check the activity of genes. An active repressor can be made inactive by adding inducer, while an inactive repressor can be made active by adding a co-repressor.
A hypothesis to explain the induction and repression of enzyme synthesis was first proposed by Jacob and Monod. The scheme proposed by them is called Operon Model.
This consists of the components:
(i) Structural genes
(ii) Promoter genes
(iii) Operator genes
(iv) Regulator genes
(v) Effector or inducer
These are directly concerned with the synthesis of cellular proteins. They produce the mRNAs through transcription and determine the sequence of amino acids in the synthesized proteins. All the structural genes under an operon may form one long poiycistronic or polygenic mRNA molecule.
This is located adjacent to the structural gene. It determines whether the structural genes are to be repressed by the repressor protein, a product of regulator gene. The operator gene is the site of binding of the repressor protein, the latter binds to the operator forming an operator-repressor complex. When the repressor binds to the operator, transcription of the structural genes cannot occur.
These genes synthesize repressor. Repressor may be either an active repressor or an inactive repressor. Repressor protein has one active site for operator recognition and other active site for inducer. In absence of an inducer protein, the repressor binds to the operator gene and blocks the path of RNA polymerase. Thus the structural genes are unable to transcribe mRNA and consequently protein synthesis does not occur.
In presence of an inducer, the repressor protein binds to the inducer to form an inducer-repressor complex. The repressor when binds with inducer undergoes a change and becomes ineffective and as a result it cannot bind to the operator gene and the protein synthesis is possible.
The actual site of transcription initiation is known as promoter gene which lies to the left of the operator gene. It is believed that RNA polymerase binds to and moves from the promoter site.
Effector or Inducer:
Effector is a small molecule (sugar or amino acid) that can be linked to a regulator protein and will determine whether repressor will bind the operator or not. In the inducible operon, these effector molecules are called inducer. In repressible operon, these effector molecules are called co-repressor.
The best known operon is the lac operon. The lac operon exercises both positive and negative control. Negative control is in the sense that the operon is normally “on” but is kept “off” by the regulator gene, i.e., the genes are not allowed to express unless required.
The lac repressor exercises negative control. Positive control is that in which the regulator gene will stimulate the production of the enzyme. Catabolite activator protein (CAP) facilitates transcription, so it exercises positive control. Two unique proteins are thus involved in the regulation of the lac operon which are lac repressor and CAP.
Lactose is a disaccharide molecule. In order to utilize lactose as a carbon and energy source, the lactose molecules must be transported from the extracellular environment into the ceil, and then undergo hydrolysis into glucose and galactose. These reactions are catalysed by three enzymes. The lac operon consists of three structural genes (lac Z, Y, A) which code for these three enzymes (Fig. 17.2).
lac Z gene — codes for enzyme β galactosidase which breaks lactose into galactose and glucose
lac Y gene — codes for permease which transports lactose into the cell
lac A gene — codes for transacetylase which transfer the acetyl group from acetyl CoA to galactose.
Negative Control of lac Operon:
lac repressor is synthesized through the activity of the lac I gene called the regulator gene. This repressor is an allosteric protein
(i) That can bind the lac DNA at the operator site, or
(ii) That can bind to inducer.
In the absence of inducer, DNA binding site of repressor is functional. The repressor protein binds to the DNA at the operator site of the lac locus and blocks the transcription of the lac genes by RNA polymerase. Thus lac enzyme synthesis is inhibited (Fig. 17.3A).
Lactose is not the real inducer of the lac operon. It binds to repressor to increase its affinity for operator. On the other hand, the bound protein of the inactive repressor is the allolactose. While β galactosidase breaks lactose into glucose and galactose, a side reaction changes galactose to allolactose and galactobiose.
This allolactose prevents the anti-inducing lac I lac lac effect of lactose. When the allolactose (inducer) binds to the repressor, it changes the form of DNA binding site making the repressor inactive and release from- the operator site. Thus transcription of lac genes are possible.
Positive Control of lac Operon:
It is an additional regulatory mechanism which allows the lac operon to sense the presence of glucose, an alternative and preferred energy source to lactose. If glucose and lactose are both present, cells will use up the glucose first and will not utilize energy splitting lactose into its component sugars.
The presence of glucose in the cell switches off the lac operon by a mechanism called catabolite repression which involves a regulatory protein called the catabolite activator protein (CAP). CAP binds to a DNA sequence upstream of the lac promoter and enhances binding of the RNA polymerase and transcription of the operon is enhanced (Fig. 17.3B).
CAP only binds in the presence of a derivative of ATP called cyclic adenosine monophosphate (cAMP) whose levels are influenced by glucose. The enzyme adenylate cyclase catalyzes the formation of cAMP and is inhibited by glucose. When glucose is available to the cell, adenylate cyclase is inhibited and cAMP levels are low.
Under these conditions CAP does not bind upstream of the promoter and the lac operon is transcribed at a very low level. Conversely, when glucose is low, adenylate cyclase is not inhibited, cAMP is higher and CAP binds increasing the level of transcription from the operon.
If glucose and lactose are present together, the lac operon will only be transcribed at a low level. However when the glucose is used up, catabolite repression will end and transcription from the lac operon increases allowing the available lactose to be used up.
The trp operon consists of the following components:
(i) Structural genes (trp E, D, C, B and A):
This operon contains five structural genes encoding enzymes involved in biosynthesis of the amino acid tryptophan. The genes are expressed as a single mRNA transcribed from an upstream promoter.
(ii) Promoter gene (trp P):
It is the promoter region which is the binding site for RNA polymerase.
(iii) Operator gene (trp O):
It is the operator region which binds with the repressor.
(iv) Leader gene (trp L):
It is the leader region which is made of 162 nucleotides prior to the first structural gene trp E. It has four regions, region 1 has the codon for tryptophan, region 2, 3 and 4 regulate the mRNA synthesis of the structural genes.
Expression of the operon is regulated by the level of tryptophan in the cell (Fig. 17.4). A regulatory gene upstream of the trp operon encodes a protein called the trp repressor. This protein binds a DNA sequence called the trp operator which lies just downstream of the trp promoter partly overlapping it.
When tryptophan is present in the cell it binds to the trp repressor protein enabling it to bind the trp operator sequence, obstructing binding of the RNA polymerase to the trp promoter and preventing transcription of the operon.
In the absence of tryptophan, the trp repressor is incapable of binding the trp operator and transcription of the operon proceeds. Tryptophan, the end product of the enzymes encoded by the trp operon, thus acts as a co-repressor with the trp repressor protein and inhibits its own synthesis by end product inhibition.
Attenuation is an alternative regulatory mechanism that allows fine adjustment of expression of the trp operon and other operons (phe, his, leu, thr operon). The transcribed mRNA sequence between the trp promoter and the first trp gene are capable of forming either a large stem-loop structure that does not influence transcription or a smaller stem loop which acts as transcription terminator (Fig. 17.5).
The relative position of the sequences does not allow the formation of both stem-loops at a time. Attenuation depends on the fact that transcription and translation are linked, i.e., ribosomes attach to mRNAs as they are being transcribed and begin translating them into protein.
Binding of ribosomes to the trp mRNA influences which of the two stem-loops can form and so determines whether termination occurs or not (Fig. 17.5).
A short coding region upstream of the stem-loop region contains tryptophan codons which is translated before the structural genes. When tryptophan levels are adequate, RNA polymerase transcribes the leader region closely followed by a ribosome which prevents formation of the larger stem-loop, allowing the terminator loop to form ending transcription.
If tryptophan is lacking, transcription is initiated, but not subsequently terminated because the ribosome is stalled, the RNA polymerase moves ahead and the large stem-loop forms. Formation of the terminator loop is blocked and transcription of the operon proceeds. When tryptophan present at intermediate levels, some transcripts will terminate and others not.
Attenuation thus allows the cell to synthesize tryptophan according to its exact requirements. Overall, the trp repressor determines whether the operon is switched on or off and attenuation determines how efficiently it is transcribed.
The sequence of the mRNA suggests that ribosome stalling influences termination at the attenuator. The ability of the ribosome to proceed through the leader region may control transition between these structures. The structure determines whether the mRNA can provide the features needed for termination or not.
When tryptophan is present, ribosomes are able to synthesize the leader peptide. They will continue along the leader section of the mRNA to the UGA codon, which lies between regions 1 and 2. By progressing to this point, the ribosomes extend over region 2 and prevent it from base pairing.
The result is that region 3 is available to base pair with region 4, generating the terminator hairpin. Under these conditions, therefore, RNA polymerase terminates at the attenuator.
However, when there is no tryptophan, ribosomes initiate translation of the leader peptide but stall at the trp codons which is at the region 1. Thus the region 1 cannot base pair with region 2. If this happens, even while the mRNA itself is being synthesized, region 2 and 3 will be base- paired before region 4 has been transcribed.
This compels region 4 to remain in a single stranded form. In the absence of the terminator hairpin, RNA polymerase continues transcription past the attenuator.
The ara (arabinose) operon of F. coli contains:
(i) Three structural genes (ara A, ara B and ara D) – which encode three different enzymes (isomerase, kinase, epimerase) for metabolism of arabinose three sructuretural genes are co-transcribed on a single mRNA.
(ii) Promoter gene(PBAD)- which initiates transcription.
(iii) Regular gene (ara C)- the regulatory protein of this gene ara C.
(iv) Promoter gene (Pc)- This initiates transcription of are C.
Two promoters PBAD and Pc are situated 100 nucleotide pairs away in the same inducer region and they initiate transcription in opposite directions.
The induction of ara operon depends on the positive regulatory effects of two proteins, the ara C protein and CAP (the cAMP binding catabolite activator protein), the binding sites of these two proteins are located in a region called ara I which is situated in between the three structural genes (ara B, ara A and ara D) and the regulator gene (ara C) (Fig. 17.6A).
The ara C protein acts as a negative regulator (a repressor) of transcription of the ara B, ara A and ara D structural genes from the PBAD promoter in absence of arabinose and cyclic AMP (cAMP). But it acts as a positive regulator (an activator) of transcription of these genes from the PBAD promoter when arabinose and cAMP are present.
Depending on the presence or absence of effector molecule like arabinose and cAMP, the ara C regulatory gene product may exert either a positive or negative effect on transcription of the ara B, ara A and ara D structural genes (Fig. 17.6B).
Post-Transcriptional Regulation of Gene Expression in Prokaryotes:
Gene regulation may also occur in prokaryotes at the time of translation.
Autogenous Regulation of Translation:
There are number of examples where a protein or RNA regulates its own production. Several proteins work as repressors, bind to the ribosome binding site (or SD-Shine-Dalgarno sequence) or initiation codon of mRNA. In these cases mRNA remains intact but cannot be translated. There are some other systems where mRNA may be degraded by the binding of protein on the short specific sequences of mRNA.
Regulation by Anti-sense RNA:
Translational control of protein synthesis can be exercised by using RNA which is complementary to mRNA, these complementary RNA will form RNA- mRNA hybrids and prevent mRNA from being translated. These kind of RNAs are called anti- sense RNA or micRNA (mic = mRNA interfering complementary RNA).
Repression of Translation:
Repression of translation occurs by the following ways:
(a) A repressor-effector molecule may recognise and bind to a specific sequence or to a specific secondary structure (involving SD region and AUG codon), thus blocking initiation of translation through blocking of the ribosomal binding region.
(b) A repressor-effector molecule may bind to an operator (not involving SD region and AUG codon) thus stabilizing an inhibitory mRNA secondary structure.
(c) An effector molecule (an endonuclease) can inhibit initiation of translation by endonucleolytic cleavage of SD region.
Activation of Translation:
Some positive effectors or activators cause activation of translation by destabilizing the inhibitory secondary structures in mRNA either through simple binding or by endonucleolytic cleavage. Translation of certain genes may be influenced by certain other genes – the phenomenon is called translational coupling.
In some cases, the end product of a particular biosynthetic pathway gets accumulated and this accumulation may stop further synthesis of this substance. The end product acts through allosteric transformation of the first enzyme of biosynthetic pathway (Fig. 17.7).