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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 minimalglucose 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 Us 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 enzymes 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-160170--> next ATG-at+1for 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-//ATGTranslated Leader: M.T.R.V.Q.F.K.H.H.H.H.H.H.H.P.D---terM


E.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 3ends
Trp
-14- MKAIFVLKGWWRTS
Phe-A
-16- MKHIPFFFAFFFTFP
His
-16- MTRVQFKHHHHHHHPD

Thr
Leu
Ilv

-21- MKRISTTITTTITITTQNGAG
-28- MSHIVRFTGLLLNAFIVRGRPVGGIQH
-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 transferse
F. 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
Gene-1(G)

Enzyme
Enzyme -1

Gene-2(I)

Enzyme-2

Gene-3(I)
Gene-4(A)
Gene-5(H)
Gene-6(F)
Gene-7(B)
Gene-8(C)
Gene-9(B)
Gene-10(D)

Enzyme-3
Enzyme-4
Enzyme-5
Enzyme-6
Enzyme-7
Enzyme-8
Enzyme-9
Enzyme-10

Name of the enzyme


PrPP
pyro
phosphorylase
Pr-AMP pyro phospho
hydrolase
Hydrolase
Isomerase
Amido transferase
ribotide cyclase
IGP dehydrase
IAP transaminase
Hp phosphotase
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 signicantly
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 signicantly 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 modied 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 signicantly 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 and
AGA 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 efciency 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.