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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).
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.
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.
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.
Thr
Leu
Ilv
-21- MKRISTTITTTITITTQNGAG
-28- MSHIVRFTGLLLNAFIVRGRPVGGIQH
-32- MTALLRVISLVVISVVVIIIPPCGALGRGKA
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
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