Bacteriophage lambda vehicle for the direct cloning of Escherichia coli promoter DNA sequences: feedback regulation of the rplJL-rpoBC operon.

Abstract

ABSIRA4CA derivative of bacteriophage X, X 21, has been constructed and used for the cloning of Escherichia coli DNA fragments carrying promoters. Phaage X 21 lacks the lac promoter-operator and can accept DNA fragments up to 9.8 kilobases in size at a unique HindIII restriction endonuclease site adjacent to lacZ -Recombinant phage that carry promoters are readily identified by their expression of lacZ Lysogens of these phages in strains harboring a deletion of the chromosomal lac operon are capable of growth on lactose as sole carbon source and can be used to study some of the regulatory signals that act upon the cloned promoter. In Promoter regions and adjacent sequences are of prime importance in understanding the molecular mechanisms of gene regulation. The fundamental problem of recognizing and defining promoter sites has been approached'through the isolation of up-or down-promoter mutants and the analysis of deletions or operon fusions (1-5). However, for those operons that specify products not readily' detectable, it is difficult to measure promoter activity or to select promoter mutants. This applies to a large group of essential genes, such as those for ribosomal proteins (r-proteins) and rRNA' whose study would be more expeditious were it not for the tedious procedures necessary for quantitation of their gene products. One way of circumventing these difficulties is to devise a means of fusing promoters to promoterless structural genes whose products are conveniently measured, such as lacZ (3). With the available increasingly detailed catalogue of promoter regions determined with restriction enzymes, it is now possible to extend this approach by in vitro construction of cloning vehicles. These would be of general use in the direct isolation of promoter fragments (6-8). MATERIALS AND METHODSBacterial and Phage Strains. The Escherichia coli K-12 strains were from our collection: NF1531 F-minA -minBthi-/pEH1 (9), JF1598 HfrH A(lac)X74 thi-relAl, JF1708 F-thr-A(lac)X74 his pyrE-argHjfucr rpsL31 this-, JF2171 F-A(lac)X74 trpE9851 rpsL.31 rho-4 thi-(10). Bacteriophage A p123(209) was obtained from M. Casadaban (3), A 540 from N. Murray (11), and X drifdl8 from J. Kirschbaum (12).Media, Phage Growth and Purification, and DNA Preparation. M9 minimal medium (13) was used and supplemented with 50Ug of L amino acids per ml and 0.4% carbon source. NZ amine (Humko, Norwich, NY) medium described by Blattner et al. (14) was used for growth of phage. Cells for phage A infection were grown in L broth (15) containing 0.4% maltose.5-Bromo-4-chloro-3-indolyl-f3-D-galactosidase (XG) (Sigma) was used at a concentration of 40 ,g/ml.A phage carrying the S7 allele were grown and purified as described (16). All other X phage were grown by the "PDS" method and purified as described by Blattner et al. (14). Phage DNA was purified as described (17) 20 Al of NaDodSO4 sample buffer (9). Extracts were prepared by boiling for 5 min and were then resolved on one-dimensional 10-20% polyacrylamide/NaDodSO4 gradient slab gels according t...

Most RNAs regulating ribosomal protein biosynthesis in Escherichia coli are narrowly distributed to Gammaproteobacteria

“…In addition, examples were identified in Tenericutes, Thermotogae, Aquificae and Deinococcus-Thermus. The RNA is responsible for regulating rplJ and rplL in response to the L10(L12) 4 complex (44,45) (Figure 1). The minimal binding site identified using phylogenetic analyses and in vitro assays is characterized by a kink-turn motif followed by a bulged cytosine in the non-canonical stem (20).…”

Relative activities of the transcriptional regulatory sites in the rplKAJLrpoBC gene cluster of Escherichia coli

“…atn + rpoBp. In addition to an attenuator, several experiments have suggested that a weak promoter is also located in the rpIL-rpoB intercistronic region (2,5,20,28). Thus, to determine whether the rpoBC genes are transcribed at a significant level from a promoter in this region, the DNA encompassed by the EcoRI sites in rpIL and rpoB was joined to lacZ (Fig.…”

“…380 nucleotides upstream of rpli (37,39). However, when segments of the operon were analyzed either by cloning them upstream of lacZ on a plasmid (2,5,21) or transducing phage (20) or directly by cloning onto a lambda transducing phage and examining the synthesis of the gene products in the UVirradiated cell system (28), it was suggested that there were internal promoters preceding rplL and rpoB. Most of these experiments indicated that these promoters were relatively weak.…”

“…Initial experiments demonstrated that the genes for the 1B and 1' subunits of RNA polymerase are cotranscribed with at least the rplJ and rplL ribosomal protein genes from a promoter upstream of rplJ (rpUp) (26,33,42). A variety of experiments subsequently suggested that there are also two internal promoters within rplJLrpoBC, one preceding rplL (rplLp) and the other before rpoB (rpoBp) (20,25,26,28,33). In addition, a transcriptional attenuator (atn) was located between rplL and rpoB and accounts for the lower frequency of rpoBC transcription compared with that of rplJL (5,6,14,26).…”

In vitro synthesis of the first dipeptide of the beta subunit of Escherichia coli RNA polymerase.

“…Although the inhibition offMet-Val synthesis by RNA polymerase with Arifdl8 DNA as template is somewhat greater than that with pNF1337 as template, a similar overall effect of RNA polymerase is observed. Maximum synthesis of fMet-Val is seen with 2-5 jig ofRNA polymerase and significant.inhibition is seen with [10][11][12][13][14][15] ,ug of RNA polymerase. As a control for these experiments, expression of the L12 gene was determined by measuring the formation of fMet-Ser (using Ser-tRNASer) under similar conditions.…”