His Operon (Molecular Biology)

Energy equivalent to about 41 ATP molecules is required to synthesize one molecule

of the amino acid histidine (1). The considerable metabolic cost of histidine biosynthesis

presumably accounts for the evolution of multiple strategies to regulate the rate of

synthesis of the amino acid in response to environmental changes. Checkpoints regulate

both the flow of intermediates through the biosynthetic pathway and the amounts of

histidine-biosynthetic enzymes present. Expression of the genes for these enzymes is

regulated in bacterial cells by mechanisms that are both general (metabolic regulation,

elongation control) and specific (attenuation control, segmental stabilization of the

distal part of the messenger RNA).

1. Structural organization of the operon

In Escherichia coli and Salmonella typhimurium the enzymes responsible for the

biosynthesis of histidine are encoded by eight genes tightly clustered in a single, large

operon (his operon). In both species, transcription produces a single polycistronic

mRNA about 7300 nucleotides long, extending from a primary promoter (hisp1) to a

Rho-independent terminator. Two weak internal promoters, hisp2 and hisp3, are located

within the hisC and hisF genes, respectively. The structural organization of the operon

is essentially the same in the two species, and in both the translational stop codon of

each cistron overlaps the translational initiation codon of the downstream cistron (2).

This organization allows ribosomes to initiate the translation of a new cistron without

moving away from the mRNA after terminating translation of the preceding one. Such a

translational coupling mechanism probably guarantees equimolar synthesis of the

corresponding gene products (3).

2. Control of transcription initiation and elongation

Transcription of the his operon is about four-fold more efficient in bacteria growing in

minimal-glucose medium than when growing in rich medium. This form of control,

called metabolic regulation, adjusts the expression of the operon to the amino acid

supply in the cell. It is mediated by the "alarmone" guanosine 5′-diphosphate 3′-

diphosphate (ppGpp), which is the effector of the stringent response (see Stringency).

The alarmone regulates the his operon positively by stimulating the primary promoter

hisp1 under conditions of moderate amino acid starvation (4).

In addition to this general metabolic control, his operon transcription is specifically

regulated by attenuation of transcription, a mechanism in which a regulatory element,

located upstream of the first structural gene of the cluster, modulates the level of

expression of the histidine biosynthetic enzymes in response to the intracellular levels

of charged histidyl-transfer RNA, His-tRNA His (see Transfer RNA) (5). The his-specific

regulatory element is transcribed in a 180-nucleotide RNA leader, which exhibits two

prominent features: (i) a 16-residue coding sequence including seven consecutive

codons specifying histidine, and (ii) overlapping regions of dyad symmetry capable of

folding into mutually exclusive, alternative secondary structures that signal either

transcription termination or antitermination (6, 7). Six RNA segments are involved in

base pairing (Fig. 1 A to F) and the stem-loop structure formed by the E and F RNA

regions, plus the adjacent run of uridylate residues, constitutes the attenuator, a strong

Rho-independent transcription terminator (Fig 1). Translational control of his operon

transcription is determined by ribosome occupancy of the leader RNA, which in turn

depends, given the peculiar composition of the his leader peptide, on the availability of

His-tRNA His. High levels of His-tRNAHis allow rapid movement of ribosomes up to the

B segment; in this case, formation of the C:D and E:F stem-loop structures will result in

premature transcription termination (Fig. 1, Attenuation). In the presence of low levels

of charged tRNAHis, ribosomes stall at the consecutive histidine codons of the leader

peptide and prevent the A:B pairing by masking the A segment. Base pairing between

the B and C and between the D and E RNA regions prevents formation of the attenuator

and determines the antitermination conformation (Fig. 1, Transcription). In the case of

severe limitation of the intracellular pool of all charged tRNAs, translation of the leader

peptide fails to initiate: under these conditions, the A:B, C:D and E:F stem-loop

structures form sequentially, producing a strong transcription termination (Fig. 1,

Superattenuation). RNA polymerase pauses after synthesis of the first RNA hairpin

(A:B). This pausing is believed to synchronize transcription and translation of the leader

region by halting the elongating RNA polymerase until a ribosome starts translation of

the leader peptide (8). The pause hairpin (Fig. 1) is the only portion of the structure

thought to form when RNA polymerase resides at the pause site.

Figure 1. Regulation of translation of the his operon messenger RNA. Top: the

nucleotide sequence of the leader region typhimurium from the transcription

initiation site (+1) to the first structural gene, hisG. Brackets above the

nucleotide se< segments (A to F) capable of forming alternative, mutually

exclusive secondary structures. The convergent arrows indica structure

required for transcriptional pausing at the downstream site indicated by a

vertical arrow (see text). The amino a peptide and of the amino-terminal region

of hisG are shown. Bottom: Schematic representation of the different conforma

(see text). The run of U’s at the 3′ end indicates the formation of the terminator

hairpin E:F. The position of the terminati (UAG) in each RNA configuration is

also indicated.

Because the absolute amount of charged tRNAHis controls the level of his

attenuation (5), mutants exhibiting high his operon expression contain defects in

tRNA His biosynthesis, aminoacylation with histidine, or tRNA His modification and

processing. The hisR gene encodes the single cellular tRNAHis; and mutations in the

hisR promoter reduce the total cellular content of tRNAHis molecules by about 50% and

thereby cause increased readthrough transcription of the his attenuator (9). The hisS

gene encodes histidyl-aminoacyl tRNA synthetase, which aminoacylates tRNA His

molecules with histidine. Mutations that lower the activity of the histidyl-tRNA

synthetase or decrease the enzyme’s affinity for histidine, tRNAHis, or ATP, affect the

level of his attenuation by reducing the percentage of tRNAHis molecules charged with

histidine (10). The hisT gene encodes pseudouridine synthase I, which catalyzes the

formation of pseudouridine residues in the anticodon region of several tRNA species,

including tRNAHis. Although the undermodified tRNA His molecules are charged with

histidine to the same extent as in wild-type strains, transcription termination at the his

attenuator is greatly decreased, because the slow rate of translation of the consecutive

histidine codons causes stalling of ribosomes (11).

The overall contribution of the internal promoter hisp2 to the expression of the distal

genes of the operon is negligible when transcription proceeds from hisp1, because hisp2

is inhibited by transcription readthrough, a phenomenon known as promoter occlusion

(12). hisp2 is also subjected to metabolic regulation, although to a lesser extent than

hisp1.

Elongation of the his-mRNA is modulated by a non-specific mechanism operating at the

level of intracistronic transcription termination elements (TTEs) (13). These elements

consist of cytosine-rich and guanosine-poor RNA regions and are the binding-activation

sites of the transcription-termination Rho factor, which is responsible for polar effects in

polycistronic operons (14). A premature arrest of translation, produced by nonsense

mutations, favors the binding of Rho to the TTE on the nascent transcript. The

subsequent interaction of Rho with the elongating RNA polymerase causes a premature

release of transcripts. Polarity results, with reduced expression of the genes located

downstream from the TTE.

3. Decay and Segmental Stabilization of the his-mRNA

The primary 7300-nucleotide his-mRNA has a half-life of about 3 minutes in cells

growing in minimal-glucose medium and is degraded in a net 5′ ^ 3′ direction. Three

major processed species, 6300, 5000, and 3900 nucleotides long, encompassing the last

seven, six, and five cistrons, respectively, are generated in the decay process (12). The

6300- and the 5000-nucleotide RNAs, which have half-lives of 5 and 6 minutes,

respectively, have heterogeneous 5′ ends generated by ribonuclease E cleavage (see

RNA Degradation In Vitro). The 3900-nucleotide processed RNA species has a unique 5′

end and an uncommon stability, having a half-life of about 15 minutes. This RNA species

is generated by specific processing events requiring sequential cleavages by two

different endonucleases. RNase E triggers the process by cleaving at a major target site

located in the hisC cistron, 620 nucleotides upstream of the 5′ end of the processed

species. Subsequently, ribonuclease P cleaves the processed RNA species generated by

RNase E at a site located 76 nucleotides upstream of the start codon of the hisB cistron,

producing the mature 5′ end. The observation that the RNase P-catalyzed reaction

requires the presence of ribosomes suggests that translation of the hisB cistron might

favor formation of the structure recognized by RNase P (15).

 Regulation of Histidine Operon: 

Histidine is an important and an essential amino acid.  It is synthesized in cells using an elaborate biochemical steps; starting from Phospho ribosyl pyrophosphate (PrPP) to L Histidine.  It requires ten steps and ten enzymes, and it means ten genes. Interestingly most of the enzymes are monomers.   All the genes are clustered into one operon under the control of one promoter-operator.  This type of organization is found, in both E.coli and S.typhimurium. S.typhimurium  P-O  genes--->P1-o-> G(1)-D(10)-C(8)-p2-B(7)-H(5)-A(4)-F(6)-p3-I(3)-E(2)  (-)35---(-10)p.box-(1)+1>pppAUC----leader-----20ATG----uuuTer-160-170--> next ATG-at+1—for the first Gene in the histidine operon---- Numerical 1 to 10 indicates the steps in biochermical pathway in the synthesis of histidine.  But the order of genes in the operon are in the same way as the genes involved in synthesis.Transcription starts at +1 at and ATG in the  leader starts at 20 and the leader terminates at +160-170—(leader sequence),and at 228 another ATG for histidine operon starts. This region acts as attenuator sequence.                    ---(35)--(-10)TAGGTTA-+1ATGAAATG--//--GGCTTTTT-ter-//ATG- Translated Leader: M.T.R.V.Q.F.K.H.H.H.H.H.H.H.P.D---ter—ME.coli K12: Histidine operon cistrons sequence I-P-O>--E-I-F-A-H-F-B-C-D-G t/t The 5’ leader sequence also contain similar attenuator sequences as found in Solmonells typhimurium operon.   

The leader sequence has 4 blocks with intra strand complementarity, thus they can form 4 stem loop structure, such as 1,2,3 and 4.When His is low or absent translation stops at H codons, and 2 and 3 block base pair, thus allow transcription to continue.  If His is present translation proceeds beyond his codons and terminates in the second loop, this provides the formation of 3 and 4 form base pairing, which generates transcription terminator stem loop with uuuuu 3’endsTrp                        -14-   MKAIFVLKGWWRTSPhe-A         -16-    MKHIPFFFAFFFTFPHis              -16-    MTRVQFKHHHHHHHPDThr              -21-    MKRISTTITTTITITTQNGAGLeu             -28-    MSHIVRFTGLLLNAFIVRGRPVGGIQHIlv               -32-    MTALLRVISLVVISVVVIIIPPCGALGRGKA 

 The pathway shown is of E.col K12 

Enzymes in sequence:G. PRPP –ATP pyrophosphorylase,E.  PR-AMP pyrophospho hydrolase,I.  Hydrolase,A. Isomerase,H. Amido transferseF. Cyclase,B. IGP dehydrase,C. IAP transaminase,B. HP phophotase,D. Histidinol dehydrogenase   These are the sequence of reactions.  In structural organization of individual gene in the operon, the first gene in the operon is  gene-2 and the last is gene-1. 

Gene Enzyme Name of the enzyme

Gene-1(G) Enzyme -1 PrPP pyro phosphorylase

Gene-2(I) Enzyme-2 Pr-AMP pyro phospho hydrolase

Gene-3(I) Enzyme-3 HydrolaseGene-4(A) Enzyme-4 IsomeraseGene-5(H) Enzyme-5 Amido

transferaseGene-6(F) Enzyme-6 ribotide cyclaseGene-7(B) Enzyme-7 IGP dehydraseGene-8(C) Enzyme-8 IAP transaminaseGene-9(B) Enzyme-9 Hp phosphotaseGene-10(D) Enzyme-10 Histidinol

dehydrogenase  

The Histidine operon is regulated in the fashion of Tryptophan operon by attenuator mechanism.  Before the start of the first cistron the transcript has a long leader sequence, which can generate two stem loop structure, one of which contains stem

loop with UUUU sequences.  This develops when Histidine present and the ribosome progresses through Histidine codons and stops at a terminator codon, this generates terminator stem loop.  But when Histidine is absent the translating ribosome stops at his codons for the lack of his carrying tRNA, this generates hair-pin loop between the 2ndand the 3rd part of the leader sequence, thus transcription is not terminated.  The attenuator mechanism is more or less similar to that of Tryptophan operon.

Implications of codon bias for molecular biologists

codon bias- The frequency with which different codons are used varies significantly between different organisms and between proteins expressed at high or low levels within the same organism.The frequencies with which different codons are used vary significantly between different organisms and between proteins within the same organism. This is referred to as codon bias.

The bottom line for wobble base pairing and modified bases in tRNAs is that there are multiple ways to construct a set of tRNAs able to recognize all the 61 codons. A particular codon family is read by tRNAs with different anticodons in different organisms. The frequencies with which different codons are used vary significantly between different organisms and between proteins expressed at high or low levels within the same organism. This is referred to as codon bias. For example, mammalian genes commonly use AGG andAGA codons for arginine, whereas these are very rarely used in Escherichia coli. E. coli is a bacterium often used for expression of recombinant human proteins. Correlating with this observation, in E. coli, the tRNAArg that reads the infrequently used AGG and AGA codons for arginine is present only at very low levels. The expression of functional proteins in eterologous hosts (i.e. hosts of a different species), is a cornerstone of molecular biology research. Codon bias can have a major impact on the efficiency of expression of proteins if they contain codons that are rarely used in the desired host. Notably, Tetrahymena, the ciliate that played an important role in the discovery of telomerase (see Section 6.9), possesses tRNAs that read the canonical stop codons UAA and UAG as glutamine (Gln), making these genes impossible to express heterologously without some type of redesign strategy of the gene or host.