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The Leader Mrna of the Histidine Attenuator Region Resembles Trnahis

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The Leader Mrna of the Histidine Attenuator Region Resembles Trnahis Proc. Natl. Acad. Sci. USA Vol. 80, pp. 5240-5242, September 1983 Biochemistry The leader mRNA of the histidine attenuator region resembles tRNAHiS: Possible general regulatory implications (tRNA-modifying enzymes/RNA secondary structure/prokaryotic and eukaryotic regulation) BRUCE N. AMES, TIMOTHY H. TSANG, MARTIN BUCK, AND MICHAEL F. CHRISTMAN Department of Biochemistry, University of California, Berkeley, California 94720 Contributed by Bruce N. Ames, May 23, 1983 ABSTRACT The leader region of the mRNA of the his op- codons present in the leader peptide and thus allows the anti- eron is involved in regulating the frequency of transcription ter- attenuator stem-loops to form (3, 14). mination through attenuation and therefore expression of the his tRNAHiS and the his Leader mRNA Are Homologous. The structural genes. We now report that the his leader mRNA has a leader mRNA sequence of the his operon (7) and the sequence remarkable sequence homology with the tRNAHU molecule. Of of tRNAHiS (12), which plays a crucial role in regulating his op- the 75 nucleotides forming tRNAHIS (not counting the -CCA tail), eron expression, have been published for some time. We have 45 are homologous to nucleotide sequences found in the his leader now found a remarkable degree of homology between the two mRNA. This homology extends to secondary structures which can RNA molecules. As shown in Fig. 1, where regions of homol- form in the leader mRNA. The stems and loops of tRNAHiS are are the RNA of the his thus related to those of the his leader mRNA which play a critical ogy boxed, leader biosynthetic operon role in regulating expression of the his operon through attenua- can be depicted in a secondary structure similar to the con- tion. Many proteins that bind tRNAHiS thus might bind to the sim- ventional tRNA cloverleaf secondary structure. Of the 75 nu- ilar structures found in the his leader mRNA and influence reg- cleotides forming tRNAHiS (not counting the -CCA tail), 45 are ulation by favoring the attenuator or anti-attenuator configuration. homologous to those in the his leader mRNA. Virtually all of These include tRNA-modifying enzymes, the histidyl-tRNA syn- the anticodon loop and stem and the TqiC stem of tRNAHis are thetase, and the hisG enzyme. The significance of similar struc- found in the his leader RNA. Considerable homology to the tures in other regulatory systems is discussed, particularly in re- dihydrouridine stem and loop and the TqiC stem and loop are lation to the role of tRNA-modifying enzymes as important reg- also found in the leader RNA. The function in attenuation of the ulatory molecules in both prokaryotes and eukaryotes. small stem and loop preceding the histidine attenuator has been unclear: it now seems reasonable that its role may be related Attenuation. The attenuator mechanism of transcriptional to its homology with the TqiC stem and loop of tRNAH1S (see regulation has been described for the tryptophan (1, 2) and his- discussion below). The leader mRNA has been pictured in the tidine (3-7) operons and for several other (2, 8-10) amino acid form that retains the attenuator. When the leader mRNA is in biosynthetic operons in Escherichia coli and Salmonella ty- the alternate (anti-attenuator) conformation there is some ho- phimurium. The attenuator is a stem-and-loop structure that mology with tRNAHiS-e.g., the m7G-containing variable loop- forms in the leader region of the mRNA transcript and prevents but little overall homology. These homologies are present in further transcription into the structural genes of the operon. both E. coli and S. typhimurium: these organisms have identical Transcription of the structural genes can only occur under con- tRNAHiS sequences (12) and, except for two bases that are out- ditions whereby alternate stem-loops in the leader region of the side of the homologous regions (3, 6), they have identical se- mRNA preclude the formation of the attenuator stem-loop. In quences in the histidine mRNA attenuation region. We have the histidine operon, this alternate RNA (anti-attenuator) con- looked for homologies between the sequences of the tRNA and figuration is favored by the shortage of charged tRNAH1S, the mRNA leader of trp, phe, thr, leu, ilv and have seen some, but principal regulatory input, resulting in a slow translation of a none of these is as striking as for his. region of the leader mRNA containing seven histidine codons The remarkable sequence and structural similarities be- and coding for a small histidine-rich peptide (3-7). Many other tween the his leader RNA and its cognate tRNA leads us to sug- amino acid operons contain analogous control regions (2, 8-10). gest that proteins that interact with tRNAHis such as modi- tRNAHiS Modification and Regulation. The realization that fying enzymes, histidyl-tRNA synthetase, the hisG enzyme, the degree of charging of tRNAHiS was the primary physiolog- tRNA processing enzymes, ribosomal proteins, and elongation ical input regulating the transcription of the his operon came factors-also could interact with similar sites in the leader mRNA. from studies on the various mutants defective in the control of Such RNA-protein interactions might provide a considerable histidine biosynthesis (11). These studies also uncovered a sec- degree of regulatory input for coupling his operon expression ond possible mechanism for a physiological input into the con- to metabolic stresses and the histidine needs of the bacterial trol of his operon expression: the degree of modification of cell. Such interactions also add a new dimension in thinking pseudouridine at positions 38 and 39 in tRNAHiS (12). This mod- about regulatory mechanisms as discussed below. ification was shown to be catalyzed by the hisT gene product, tRNA-Modifying Enzymes and Regulatory Mechanisms in a tRNA pseudouridylating enzyme (13). The undermodified Both Prokaryotes and Eukaryotes. It has been postulated that tRNA, even though fully charged, causes a derepression of the many of the modified nucleosides in tRNA represent regula- histidine biosynthetic pathway. Pseudouridine-deficient His- tory signals (13, 15, 16; unpublished data). More than 30 mod- tRNAHiS appears to slow translation of the seven contiguous his ifications and modifying enzymes exist in S. typhimurium and E. coli (17, 18). The number of modified nucleosides in higher The publication costs of this article were defrayed in part by page charge organisms is even greater (17). A specific metabolic stress can payment. This article must therefore be hereby marked "advertise- affect a tRNA-modifying enzyme to result in a lack of modi- ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. fication in a family of tRNAs (15, 16; unpublished data). Spe- 5240 Downloaded by guest on September 23, 2021 Biochemishy: Ames et aL Proc. Natl. Acad. Sci. USA 80 (1983) 5241 pppAUCAAAUGAAUAAGCAUUC A u rno: start GOAOOOObAA GcG A A U ACUGCA UA A-OH AACUUGC UA C C U c ACCEPTOR u CATTENUATOR STEM A GCCSKiTEM UA U-OH A U FG G-C A CA Uj U-A C-GA-U ACCA ro ..u *mdA-U U CG C G< C 4 G-G:C * bmodified Q- t RNAHIs HISTIDINE LEADER mRNA FIG. 1. Sequence and structural homologies between tRNA"iS and the histidine leader mRNA of S. typhimurium. (* indicates modified nu- cleoside; parent nucleoside is shown.) cific differences in modification can lead to altered transcrip- presence of a leader peptide in mRNA could also allow a purely tion through slowed translation of a leader peptide influencing translational control through ribosome position influencing sec- attenuator formation (14, 16). The sequence similarity of his ondary structures or blocking initiation sites (see the 1967 Mar- leader mRNA and tRNAHiS suggests additional mechanisms for tin-Ames model in ref. 14). An example of a leader peptide the control of his and other operons. Four enzymes that modify with a series of leucine codons has been found in front of a leu- tRNAHiS might interact with the his leader RNA. Possible mod- cine biosynthetic structural gene in yeast and has been dis- ifications include the U-38 and U-39 pseudouridines (the mod- cussed with respect to both translational and transcriptional ification catalyzed by the hisT gene product), as well as m2A, regulation (23). In addition, several animal viruses have se- m7G, and D. We have recently reviewed the sequence rules for quences suggesting the existence of some form of attenuation modification-enzyme recognition sites on tRNA (19). Because (reviewed in ref. 2). In the case of simian virus 40, a small pro- each modifying enzyme binds a region of RNA with a particular tein may stabilize one of two alternative conformations of the secondary structure and sequence (19), the modifying enzymes mRNA, resulting in both translational and transcriptional (pre- form a large family of proteins that could act as transducers of mature termination) effects (24). metabolic stress to regulate gene expression. Thus the modi- Histidyl tRNA Synthetase. The hisS protein has been pos- fying enzymes might bind to, or actually modify, the leader tulated to play an accessory role in histidine regulation (25). A mRNA and directly influence (i) competing stem-loop forma- high-copy-number plasmid containing the hisS gene causes de- tion and, therefore, formation or preclusion of an attenuator repression of the his operon (26). One proposed explanation is structure favoring transcription termination, (ii) translation ini- that attenuation is relieved by the excess tRNA synthetase's se- tiation (analogous to ref. 20; see also ref. 21), (iii) mRNA sta- questering tRNAHiS (7). Another possibility is that the tRNA bility, or (iv) mRNA processing. Therefore, a physiological stress synthetase binds the leader mRNA by some of the same bind- could be coupled to regulation through the modifying-enzyme ing sites it has for tRNAHiS to influence attenuator loop for- transducers.
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