Regulation of Gene Expression in Prokaryotes | Gene Regulation

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.

Operon Model:

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

Structural Gene:

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.

Operator Gene:

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.

Regulator Gene:

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.

Promoter Gene:

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.

Inducible Operon:

Lac Operon:

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.

Repressible Operon:

Trp Operon:

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:

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.

Ara Operon:

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(P_BAD)- 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 P_BAD 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 P_BAD 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 P_BAD 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.

Feedback Inhibition:

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).