153 , No. 2 Internal Promoters of the his Operon in Salmonella typhimurium MOLLY B. SCHMIDt AND JOHN R. ROTH * Biology Department , University of Utah , Salt Lake City , Utah 84112 Received 13 September 1982/Accepted 29 November 1982 Two internal promoters in the his operon of Salmonella typhimurium have been precisely mapped genetically .
The internal promoters are found in , or very close to , gene border regions in the his operon .
The his operon was examined for the presence of additional internal promoters whose transcripts were sensitive to rhomediated transcription termination and therefore had escaped detection .
No new internal promoters were found .
It is argued that the internal promoters described here are not likely to be fortuitous message start sites , but may play a physiologically important role in operon expression .
Internal promoters appear to be quite common in large bacterial operons ( 2 , 3 , 6 , 9 , 14 ) , but their role is not understood .
They could allow regulation of the operon in a noncoordinate manner , allowing different levels of gene products in the operon or temporally different expression of certain genes .
Alternatively , their major role could be to reinforce expression of downstream genes , which would otherwise be transcribed in lower amounts owing to the effects of natural polarity .
It is also possible that these promoters are physiologically unimportant and are merely fortuitously occurring sequences able to act as low-level promoters .
To approach the study of such promoters , we have accurately mapped their location in the histidine operon .
The histidine biosynthetic operon of Salmo-nella typhimurium consists of nine genes that are regulated coordinately .
All of these genes are transcribed from a primary promoter ( P1 ) , which is subject to attenuation control ( 1 , 15 , 17 ) .
Two other functional promoters exist within the his operon ( 2 , 7 ) that are capable of transcribing portions of the operon .
These internal promoters have been roughly mapped previously but have never been precisely located ( 2 , 7 , 19 , 22 ) .
The general location of the his internal promoters was first described by Atkins and Loper ( 2 ) and has been confirmed by others ( 7 , 19 , 22 ) .
Expression originating from the internal promoters was observed by eliminating the primary promoter ( P1 ) by deletion ( 2 , 7 , 22 ) or by blocking Pl transcripts with a TnJO insertion ( 19 ) .
The P2 promoter then allows expression of the hisB , H , A , F , I , and E genes .
The P2 promoter was previously mapped to a region including the last t Present address : Institute of Molecular Biology , University of Oregon , Eugene , OR 97403 .
half of the hisC gene and the first half of the hisB gene ( 2 , 20 ) .
Another internal promoter ( P3 ) allows expression of the hisI and hisE genes and can be seen when the P1 and P2 promoters are removed .
A genetic map showing the his operon and location of the internal promoters is presented in Fig. 1 .
The precision of the mapping by Atkins and Loper ( 2 ) was limited by the number of available deletions that removed the Pl or both the PI and P2 promoters ; the precision of mapping by Kleckner and co-workers ( 19 , 20 ) was limited by the number and location of available TnlO insertion mutations .
The experiments described here rely on a TnlO insertion to block transcription from the major his promoter Pt , thus allowing the presence or absence of downstream gene expression to be absolutely correlated with the presence or absence of the internal promoter .
The TnIO element itself has a promoter that is able to transcribe sequences outside of the inserted element ( 4 ) , but these TnJO-initiated transcripts are efficiently terminated by the rho-dependent termination mechanism and do not express downstream genes under the conditions used ( 4 ) .
Experimentally , expression arising from a his internal promoter is easily differentiated from expression arising from a TnJO promoter .
Genetic mapping of the first internal promoter , P2 .
Previous work on the P2 promoter has placed it within a region including the last half of the hisC gene and the first half of the hisB gene , as shown in Fig. 1 .
To refine the location of P2 , strains were constructed that have hisG8570 : : TnJO ( which blocks transcripts from Pl ) and an internal his deletion .
Strains used are listed in Table 1 .
In an otherwise wild-type strain , this hisG : : TnlO insertion prevents expression of the hisG , D , and C genes ; hisB and subsequent genes are transcribed from P2 .
If the tested his deletion removes the P2 promoter , then hisB , H , A , and F will not be expressed .
If the his deletion does not remove the P2 promoter , then hisB , H , A , and F will be expressed .
In either case , the genes hisI and E will still be expressed , owing to the P3 internal promoter .
Gene expression was measured by complementation , using a series of Escherichia coli F ' his episomes carrying various well-characterized his mutations on the episome .
Data critical to the final placement of P2 are shown in Table 2 .
It is concluded that deletion his-129 does not affect the P2 promoter , deletion his-22 removes or severely damages P2 , and deletion his-2648 damages but does not remove the promoter ( see Fig. 1 ) .
P2 promoter directs expression of hisB phosphatase .
The endpoint of his-22 is very close to the hisC-hisB gene border .
There are no hisC point mutations known to be deleted by his-22 , and there are no hisB point mutations known to lie outside deletion his-22 ( 11 ) .
The hisB enzyme is a single polypeptide encoding two distinct enzymatic functions : a phosphatase and a dehydratase .
The regions encoding these two activities are clearly separated on the hisB genetic map ; the phosphatase activity is encoded in the promoter proximal region of hisB , whereas the dehydratase activity is located in the promoter distal region , near hisH ( 13 ; T. Kohno and B. Cooper-Kohno , personal communication ) .
It has been found that a nonspecific acid phosphatase , encoded by the phoN gene , unlinked to the his operon and normally uninvolved in his biosynthesis , can substitute for the hisB phosphatase activity ( T. Kohno , manu-script in preparation ) .
Thus , mutations eliminating only the phosphatase activity of the hisB gene product are suppressed by the phoN + gene , and strains containing such hisB mutations are not detected as his auxotrophs .
Mutations in either the phoP or phoN gene abolish the suppressing nonspecific phosphatase activity ( 18 ; Kohno and Cooper-Kohno , personal communication ) .
Strains carrying either of these pho mutations can not grow on minimal-medium without a functional hisB phosphatase ( Kohno , personal communication ) .
Both hisB complementation and hisB phosphatase enzyme levels produced by the Pl or P2 promoters were measured in strains which carry either the phoP or phoN mutation .
These data are shown in Table 2 , lines 10 and 11 , and in Table 3 .
From these data , it can be concluded that P2 must direct expression of the hisB phosphatase , since strains lacking both the major his promoter ( Pl ) and the pho phosphatase retain hisB phosphatase complementation and enzyme activity .
The location of the P2 promoter must be within , or at the end of , deletion his-22 but must precede the operator-proximal phosphatase segment of the hisB gene .
This limits P2 to a tiny region including the intercistronic punctuation sites at the hisC-hisB border ( see Fig. 1 ) .
Genetic mapping of the second internal promoter , P3 .
The general location of the P3 promoter was determined by Atkins and Loper ( 2 ) and by 1 11 F P3 ' I P2 I E a 0 9 v a a 4 ¬ 228 129 97327 LI645 2648 152 FIG. 1 .
A genetic map of the his operon of S. typhimurium .
P1 , P2 , and P3 indicate the map locations of the three known promoters in the his operon .
Location of the P2 promoter Complementation of F ' T80 his episomesa D C B A F - + + + G his + + + + AHAFIE + + + IE + + + + ¬ his-129 ¬ ¬ ¬ + hisG : : TnlO his-129 hisG : : TnlO + + ¬ ¬ ¬ + + + + + + + ¬ + + + ¬ his-22 + ¬ ¬ ¬ ¬ T hisG : : TnlO his-22 hisG : : TnlO + + ¬ ¬ ¬ + + + + + + + + ¬ + + + + + his-2648 + ¬ ¬ ¬ + hisG : : TnlO his-2648 hisG : : TnlO + + + + ¬ ¬ ¬ + + + + + hisG : : TnlO phoN + -- + + + + + hisG : : TnlO phoP + -- + + + + + a Complementation data with F ' T80 his episomes with either a his + operon ( TR35 ) or a his mutation , as listed across the top of the table .
The recipient strains are grouped into isogenic sets .
The data were gathered initially by spot tests and , for any ambiguous or critical results , repeated with a full plate test .
A + indicates that good complementation was observed within 24 to 36 h at 37 °C .
A ± indicates that complementation was observed within 48 to 72 h , but that it was weaker than that scored with a + .
A T indicates that barely detectable complementation was observed after 48 to 72 h ; the growth response was never strong , even after extended incubation .
Pairs scored as-showed no detectable complementation Strain Phenotype Pi P2 rho Activity LT2 His ' + + + 2.36 TA2361 His ' , PhoN - + + -2.06 TA2362 His + , PhoP - + + -1.92 his-646 HisOGD -- + + 0.32 TT428 HisG : : TnlO - + + 0.58 TT4156 HisG : : TnJO , PhoN -- + -0.42 TT4157 HisG : : TnlO , PhoP -- + -0.48 T17416 HisOGD - , PhoN -- + -0.22 TT7417 HisOGD , PhoP -- + -0.33 his-22 HisBH -- + 0.05 TT2789 HisBH - , PhoN -- 0.0 TT2790 HisBH - , PhoP -- 0.0 a Assay levels are the average of two experiments , except in the case of TT2789 and TT2790 , which were performed only once .
Columns 3 and 4 indicate which his promoters are transcribing the hisB gene , whereas column 5 indicates the presence ( + ) or absence ( - ) of the phoN phosphatase activity .
The assays were performed on toluenized cells as described by Martin et al. ( 20a ) .
Kleckner et al. ( 19 , 20 ) , who placed P3 within the hisF gene .
To precisely map this promoter , we applied the same methods that were used to map the P2 promoter .
A TnlO insertion in either hisB , hisH , or hisA was used to block transcription from both the Pl and P2 promoters .
Normally , a strain carrying one of these TnJO insertions will complement hisI and hisE mutations , because of the P3 promoter .
These TnlO insertions were combined with deletions having endpoints within hisF .
If the deletion in question removes P3 , then the hisl and hisE genes will not be expressed ; if the deletion mutant retains P3 , then the hisI and hisE genes will be expressed and mutations in these genes will be complemented .
Table 4 shows the complementation data that test for expression of the hisI and hisE genes for the deletions critical for placing P3 .
It is concluded that deletions his-327 and his-152 remove P3 , whereas deletion his-645 leaves P3 intact ( see Fig. 1 ) .
The last two deletion intervals in hisF are genetically quite small ; if the number of point mutations is taken as an indicator of physical distance , the P3 promoter must be within the last 4 % of the hisF gene ( 11 ) .
Absence of additional internal promoters in the his operon .
One would expect that most fortuitous message starts within operons would result in transcripts which are promptly terminated by rho factor , which is known to cause termination of untranslated messages ( 8 ) .
This has recently been shown to be true in the his operon in which the outward-directed promoters of TnWO are usually not detected , owing to early rho-depen-dent termination ( 4 ) .
The only internal message starts that might be detectable are those that happened to be located so near a ribosome binding site that no rho-dependent termination site intervened ; such promoters would generate transcripts which would be protected from termination and could express distal genes .
The two promoters described here are near the ends of genes , so the possibility was entertained that they might be fortuitously occurring message start sites that were detectable owing to their location near ribosome initiation sites .
If this were the case , one would expect that many other intemal message starts might exist which are only detectable in the absence of rho factor .
We sought these cryptic message starts by looking for internal his promoters in strains carrying a rho mutation .
By making use of transductional linkage between rho and ilv , strains were constructed that contained a rho mutation ( rho-Hll [ 12 ] ) and a his deletion that removed Pl or both Pl and P2 .
The ability of these strains to express his genes was tested by complementation to determine whether Rho-strains initiated transcripts at sites other than the previously described internal promoters .
As seen in Table 5 , the complementation pattern of these deletions in Rho ' and Rho-strains is identical .
Thus , within the regions checked by this test , no additional promoters are in evidence .
The failure to find cryptic internal promoters encourages us to believe that fortuitous message starts within operons are rare and that the existence of P2 and P3 may reflect a selectively significant role for these promoters .
Several other lines of evidence the support selective importance of internal promoters .
Winkler et al. ( 22 ) have reported that the his P2 promoter is subject to ppGpp control and is activated by shift-down growth-conditions .
Thus , this promoter seems to be under metabolic control .
Evolutionary evidence supports the significance of internal promoters in both the his and trp operons .
In both S. typhimurium and E. coli , internal promoters are found within the trpD gene ( 3 , 14 ) , as well as near the hisB gene ( P2 ) ( 10 ) .
Like the his promoters described here , the trp promoter is located at the distal end of the trpD gene , near an intercistronic region .
The conservation of these internal promoters by both S. typhimurium and E. coli is striking , since DNA sequencing shows that coding sequences differ by 15 to 25 % in the trp operons of these two bacteria ( 5 , 21 ) .
It seems likely that these internal promoters have been maintained by selective pressure .
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