leu operon of Salmonella attenuation mechanism ABSTRACT The nucleotide sequence of the control region of the leu operon of Salmonella typhimurium was determined .
A prominent feature of this region is a signal for termination of transcription .
In vitro , transcription does terminate at this site , yielding a leader RNA of about 160 nucleotides as a major product .
This leader RNA is potentially translatable into a peptide containing 28 amino-acids , 4 of which are adjacent leucine residues .
Several regions of base complementarity exist within the leader , positioned such that pairing of one region precludes pairing of another .
The position of the four leucine codons relative to two regions of base complementarity suggest a model for the regulation of the leu operon similar to that proposed by Yanofsky and coworkers for the trp operon .
In addition , a third region of base complementarity was identified which , when incorporated into the model , explains why premature termination is the usual outcome when transcription is initiated in-vitro by purified RNA polymerase .
Genes specific to leucine biosynthesis are clustered on the chromosome of Salmonella typhimurium ( 1 ) and their expression is controlled by the intracellular concentration of leucine ( 2 ) .
Several classes of mutations affect expression of leu genes in a coordinate fashion .
Promoter mutations ( 3 ) eliminate expression of all four genes whereas so-called operator mutations ( 4 ) result in constitutive expression of leuA,-B , - C , and-D .
These mutations define the control region of the leu operon .
In addition , mutations unlinked to leu , in leuS and flrB , lead to constitutive synthesis of leu and some ilv gene products ( 5 ) .
The leu operon of S. typhimurium was cloned on plasmids pSC101 and pMB9 and sites at which restriction endonucleases cleave the operon were determined and correlated with a genetic map ( unpublished data ) .
In this paper we report the sequence of nucleotides in the leu operon control region .
It is apparent from the sequence and from the results of in-vitro-transcription studies that the leu operon is controlled by an attenuation mechanism similar to that described by Yanofsky and his coworkers for the trp operon ( 6 , 7 ) .
lation ( 8 ) , treatment with restriction endonucleases ( 9 ) , and separation of DNA fragments by electrophoresis ( 9 ) were as cited .
Fragments 1-6 identified by arrows in Fig. 1B were isolated as follows .
The smaller of the two fragments produced by Pst I cleavage of EcoRI5200 , Pst I-B , was cleaved with Hpa II , the ends were labeled with 32P by sequential treatment with bacterial alkaline phosphatase and polynucleotide kinase together with [ ` y-32P ] ATP ( 10 ) , and the resulting mixture was treated sequentially with Hin II and Hae II .
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electrophoresis yielded fragment 1 ( Hin II/Hpa II126 ) and fragment 2 ( Hpa II/Hae II88 ) , each labeled only at the end created by Hpa II .
Pst I-B was cleaved with Taq I , ends were labeled by means of kinase , and , after treatment with Hae III , fragments 3 ( Hae III/Taq I320 ) and 4 ( Taq I/Hae III140 ) were isolated .
Fragments 5 and 6 are identical to 3 and 4 , respectively , except that labeling was effected at 3 ' ends by means of reverse transcriptase ( a gift from J. Beard ) and [ a-32P ] dCTP ( 11 ) .
DNA sequencing was performed by the method of Maxam and Gilbert ( 12 ) with pyridinium formate for the A+G cleavage reaction ( A. Maxam , personal communication ) .
All procedures using recombinant DNA were performed under P2 conditions in accordance with the National Institutes of Health guidelines .
Conditions for in-vitro-transcription were modified slightly from those used by Lee and Yanofsky ( 6 ) .
Reaction mixtures ( total volume , 100MlI ) contained : Tris acetate ( pH 7.9 ) , 20 mM ; KC1 , 100 mM ; magnesium acetate , 4 mM ; EDTA , 100 AM ; dithiothreitol , 100 MM ; GTP , 125 AM ; ATP , 125 MM ; UTP , 75 MM ; CTP , 75 , M ; [ a-32P ] UTP , 0.12 nmol [ 186 Ci/mmol ( 1 Ci = 3.7 X 1010 becquerels ) ] ; RNA polymerase ( either New England BioLabs or a gift from J. Alegre ) , 2.0 Mg - ; and DNA , 2 Mg ( plasmid ) or 0.5 Mg ( fragment ) .
After incubation for 30 inii at 37 °C , RNA was isolated ( 6 ) , fractionated by electrophoresis on 0.4-mm-thick 8 % acrylamide gels containing 8 M urea ( 13 ) , and visualized by autoradi-ography .
RESULTS Identification of DNA Fragments Carrying the Control Region of the leu Operon .
An EcoRI-generated fragment of Salmonella DNA cloned on plasmid pMB9 ( yielding plasmid pCV12 ) carries leuABC and part of leuD togethet with a functional leu promoter and control region ( unpublished data ) .
A restriction endonuclease map and the genetic map of this fragment are shown in Fig. 1 A and D , respectively .
Correlation between the two maps was achieved by analyzing the operon from strains having deletions within leu ( detailed results to be published elsewhere ) .
Fronl a genetic analysis , one end of de-letion leu-4168 ( Fig. 1C ) lies between known promoter mutations and operator mutations ( unpublished results ) .
De-letion leu-5058 , on the other hand , extends through the promoter/operator region and just enters leuA ( it recombines with 270 of 272 mutations in leuA ) ( 14 ) .
Based upon hybridization of Hin II-and Pst I-cleaved total Salmonella DNA to labeled leu DNA by procedures developed by Southern ( 15 ) , both leu-4168 and leu-5058 end within fragment Hin II-C and to the left of the Pst I site ( Fig. 1A ) .
A more precise determination of the end point of leu-4168 was obtained by cloning the operon from the strain and comparing endonuclease-generated frag ¬ T residues between positions 191 and 197 ; to the left of this sequence and overlapping it is an 11-base-pair palindromic sequence composed predominantly of G and C residues .
These palindromic and T7 sequences together may comprise a rec-ognition element for termination of transcription ( 16 ) .
Transcription Initiated In Vitro at the leu Promoter Yields a Short RNA Transcript as a Major Product .
Plasmids pCV12 and pCV23 are identical except that the latter has promoter mutation Ieu-500 ( unpublished data ) .
Strains carrying leu-500 produce only barely detectable levels of leu enzymes ( 1 ) .
DNA from these plasmids served as templates for in-vitro transcr ` ip-and terminated at the right end of the Hae III fragment .
Several minor species of about that size were visible on the autoradiograms but their synthesis was not abolished by the leu-500 promoter mutation .
Lee and Yanofsky ( 6 ) observed that the amount of trp readthrough RNA synthesized in-vitro was dramatically increased when ITP replaced GTP .
Under conditions of in-vitro synthesis similar to theirs , the most prominent product was comparable in size to the putative readthrough RNA ( Fig. 3 , lane E ) .
There was also a minor product corresponding in size to the major species observed when reactions contained GTP .
Neither species was formed when the template contained the leu-500 mutation ( Fig. 3 , lane F ) .
These results suggest that leu RNA synthesis is initiated about 250 base pairs from the right end of the Hae III fragment ( Fig. 1B ) .
DISCUSSION The DNA molecules that were sequenced were known from genetic and restriction endonuclease analysis to contain the leu promoter and control region .
A striking feature of the derived sequence is a signal for termination of transcription ( 16 ) , hereafter referred to as the leu attenuator .
The presence of a functional attenuator was confirmed by the observation of a 165-nucleotide RNA molecule as a primary product of in-vitro-transcription .
The following evidence indicates that the synthesis of this RNA transcript was initiated at the leu promoter and terminated at the leu attenuator .
( i ) A leu promoter mutation abolished in-vitro synthesis of the transcript .
( ii ) In vitro synthesis programmed by various templates and hybridization studies demonstrated that the RNA molecule was transcribed from just the region of DNA that was sequenced .
( iii ) The major product of transcription from the Hae III fragment in the presence of ITP was about 250 nucleotides long .
Its synthesis was initiated at leuP and most likely terminated at the right end of the fragment .
This places the point of transcription initiation about 250 base pairs from the right end of the Hae III fragment , near position 35 in Fig. 2 .
Between positions 1 and 30 there is a potential region of interaction with RNA polymerase ( 17 ) .
( iv ) In the presence of GTP , synthesis of the major RNA product terminated at a point about 165 base pairs from leuP-that is , near position 195 ( Fig. 2 ) .
This corresponds to the position of the proposed attentuation site .
The strand shown in Fig. 2 is demonstrated to be the noncoding strand on the basis of the following results .
The ATG that signals initiation of translation of a-IPM synthase ( leuA gene product ) is 41 bases downstream from the attenuator site ( position 238 using the numbering system in Fig. 2 ) .
This result was deduced from additional nucleotide sequences ( data not shown ) together with the sequence of 10 amino-acids at the NH2 terminus of a-IPM synthase ( unpublished data ) .
The leader transcript was the predominant product of transcription when a Hae III fragment was used as substrate .
A putative readthrough transcript was not observed , indicating that termination of transcription was extremely efficient under the conditions of these experiments .
Presumably , termination of transcription is not so efficient in-vivo , at least not under conditions of leucine limitation in which the amount of leu mRNA is increased as much as 20-fold relative to conditions of leucine sufficiency ( ref .
A mechanism by which termination of transcription might be suppressed in-vivo has been suggested by Zurawski et al. ( 7 ) .
This mechanism focuses on certain secondary structures within the trp and phe leader RNAs that affect the frequency of termination at the respective attenuators and on the relationship of translation of the leader RNAs to transcription termination .
The leu leader contains a single AUG codon centered at position 65 , togethe codons and lag behind the polymerase .
At the point shown in Fig. 4A , protector regions have been transcribed but they are prevented from pairing by the ribosome .
However , as soon as the second half of the preemptor is transcribed , it is free to pair with its complementary region and , in so doing , prevents terminator regions from pairing ( Fig. 4A ) .
The inability to form the termination stem and loop results in continued transcription into the leu structural genes .
This circumstance , in which the ribosome pauses at the leucine codons , we believe is the only one allowing substantial transcription readthrough .
When the ribosome moves past the leucine condons ( conditions of leucine sufficiency ) , part of the preemptor is covered by the ribosome and is unavailable to preempt formation of the terminator ( Fig. 4B ) .
If the ribosome pauses near the UAA termination codon for even a fraction of a second , that is sufficient time for the termination loop to be formed ( RNA elongation rate at 37 °C is about 45 nucleotides/sec ) ( 27 ) .
On the other hand , if the ribosome separates from the RNA immediately upon reaching the termination codon , the protector regions will pair , with the result again being formation of the termination loop ( Fig. 4C ) .
This model explains why termination of transcription at the leu attenuator is so efficient during in-vitro-transcription .
This is so because in the absence of translation , the protector is the first region to pair ( Fig. 4C ) .
The physiological significance of the protector region may be to prevent derepression of the operon during limitation for an amino-acid other than leucine ( Fig. 4D ) .
It may be noted that pairing regions similar to the leu protector are present in the trp , phe , his , and thr ( 23 ) op-erons ( our analysis ) .
Preliminary results on mutants having constitutive levels of leu enzymes suggest that attenuation is the major mechanism by which leucine regulates expression of the leu operon ( unpublished data ) .
Note Added in Proof .
The unpublished data cited in the Introduction and Results are reported in ref .
Several investigators have independently developed a model for control of transcription termination that includes as a basic feature a pairing region within the leader RNA that is analogous to the protector region described here ( C. Yanofsky ; J. Roth ; personal communication ) We thank V. Vogt and J. Roberts for helpful discussions .
This research was supported by grants from the National Science Foundation ( BMS74-21987 to J.M.C. ) and the National Institutes of Health ( GM10791 to E. 3B .
K. and Al14340 to J.M.C. ) .
R.M.G. and S.R.W. were supported by National Institutes of Health Training Grant GM07273 .
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