RESUMEN

The capacity of incomplete segments of Escherichia coli lactose permease to form transport-competent complexes in vivo was further tested. Two series of mutant lacY genes were constructed. One encoded N-terminal lactose permease segments of different length. The proteins specified by the other group contained deletions of different length and location within the N-terminal region. Several pairs of such mutant proteins reconstituted active lactose transport. For certain combinations duplications of protein segments were compatible with the formation of an active carrier. Duplication of helices could also be tolerated, when part of a single polypeptide chain.

Asunto(s)

RESUMEN

Escherichia coli lactose permease mediates the proton-driven translocation of galactosides across the cytoplasmic membrane. To define regions important for membrane insertion as well as for biological function, we constructed plasmids encoding different portions of the lactose carrier. Among several lacY deletions, two were obtained that encoded mutant proteins with complementary amino acid sequences. The truncated polypeptide Y71/1 (amino acid residues 1 to 71) comprises the first two alpha-helices predicted for the intact protein, and polypeptide delta Y4-69 carries an internal deletion of this region. Regulated coexpression of these lacY-DNA segments governed by separate but identical lacOP control regions resulted in functional complementation with the following characteristics. (i) Simultaneous synthesis of both incomplete proteins restored transport activity in transport-negative cells, measured as accumulation of [14C]lactose. (ii) Under complementing conditions, but not in the absence of the smaller N-terminal protein, specific radiolabeling of the larger polypeptide by N-ethylmaleimide was prevented by substrate. (iii) The presence of the complementing N-terminal polypeptide was also required for the detection of the larger C-terminal protein by antibodies directed against the C terminus of lactose permease, indicating a stabilizing effect contributed by the smaller N-terminal fragment. Thus, coexpression of lacY mutant genes encoding two nonoverlapping portions of the lactose carrier resulted in reconstitution of a two-subunit protein in the cytoplasmic membrane exhibiting biological properties of intact lactose permease.

Asunto(s)

RESUMEN

LacY-ompA fusions, encoding the N-terminal 50, 71 or 143 residues of lactose permease, were constructed. The observed orientation of the OmpA part of each hybrid protein with respect to the plasma membrane supports current models of the N-terminus of Lac permease. Hybrids possessing the entire mature OmpA were very stable; those with only a part thereof were much less stable. Due to their in vivo stability and accessibility to antibody it is proposed that such hybrids may represent potential models to investigate the assembly pathway of lactose permease.

Asunto(s)

RESUMEN

The Escherichia coli galactose operon contains an unusual array of closely spaced binding sites for proteins governing the expression from the two physically overlapping gal promoters. Based on studies of two gal promoter-up mutants we have previously suggested RNA-polymerase-induced DNA bending of gal promoter DNA. Here we present new evidence confirming and extending this interpretation. It was obtained by the circular permutation assay of gel electrophoretic mobility [Wu and Crothers (1984), Nature, 308, 509-513] applied to three analogous series of circularly permuted fragments derived from wild-type and two promoter-up mutant DNAs. The same circularly permuted DNA fragments have further been used to study the binding of gal repressor to its operator sites by electrophoretic mobility shift and by DNase I footprinting techniques. The main results are: (i) complexes carrying repressor either exclusively at the upstream operator O1 or at the downstream operator O2 exhibit different electrophoretic mobilities; (ii) binding to either one of the operators results in protein-induced DNA bending by the criteria of the circular permutation mobility assay; and (iii) occupation of both gal operators by gal repressor does not prevent cAMP-CRP-independent binding of RNA polymerase to the gal promoters, as judged by DNase I protection and gel retardation assays. The latter finding imposes constraints on any attempt to model the regulation of gal expression by assumed DNA-protein and protein-protein interactions.

Asunto(s)

RESUMEN

Using in vitro DNA manipulations, we constructed different lacY alleles encoding mutant proteins of the Escherichia coli lactose carrier. With respect to structural models developed for lactose permease, the truncated polypeptides represent model systems containing approximately one, two, four, and five of the N-terminal membrane-spanning alpha-helices. In addition, a protein carrying a deletion of predicted helices 3 and 4 was obtained. The different proteins were radiolabeled in plasmid-bearing E. coli minicells and were found to be stably integrated into the lipid bilayer. The truncated polypeptides of 50, 71, 143, and 174 N-terminal amino acid residues resembled the wild-type protein in their solubilization characteristics, whereas the mutant protein carrying an internal deletion of amino acid residues 72 to 142 of the lactose carrier behaved differently. Minicell membrane vesicles containing truncated proteins comprising amino acid residues 1 to 143 or 1 to 174 were subjected to limited proteolysis. Upon digestion with proteases of different specificities, the same characteristic fragment that was also produced from the membrane-associated wild-type protein was found to accumulate under these conditions. It has previously been shown to contain the intact N terminus of lactose permease. This supports the idea of an independent folding and membrane insertion of this segment even in the absence of the C-terminal part of the molecule. The results suggest that the N-terminal region of the lactose permease represents a well-defined structural domain.

Asunto(s)

RESUMEN

Plasmids encoding N-terminal segments of the Escherichia coli lactose permease (also referred to as lactose carrier) have been used to analyze the biosynthesis and membrane insertion of this complex integral protein of the cytoplasmic membrane. Such truncated polypeptides were found to be stably associated with the membrane and to resemble the full-length protein with respect to their solubilization characteristics. Membrane-bound and free cytoplasmic polysomes were prepared from plasmid-bearing cells and incubated in the presence of [35S]methionine to permit completion of polypeptides initiated in vivo. Under these conditions, lactose permease was found to be radiolabeled in the fraction of membrane-bound polysomes; beta-galactosidase, used as a control, was translated almost exclusively by free polysomes. From similar experiments with N-terminal segments of lactose permease, we estimate that at most a polypeptide of 120 amino acid residues emerging from the ribosome is needed to target the nascent chain to the lipid bilayer and to mediate attachment of the ribosome to the membrane during elongation. Additional data support the idea that even shorter N-terminal sequences of 50 and 71 amino acid residues contain sufficient 'information' to provide contact with the membrane.

Asunto(s)

RESUMEN

Two mutations are described, each of which renders the Pribnow box sequence of one of the two overlapping promoters of the Escherichia coli galactose operon identical to the consensus sequence TATAAT. Both double exchanges were specifically introduced into the original context by oligonucleotide-directed mutation construction. Each of the mutant promoters exhibits a greatly enhanced capacity to form stable complexes with RNA polymerase, as judged by nuclease protection experiments and by assaying shifts of electrophoretic mobility. On the other hand, the effect of the same mutations on the rates of transcription from the two gal promoters is strikingly different. Unexpectedly, when complexed with RNA polymerase, DNA fragments carrying one of the two double exchanges were found to differ from each other as well as from the corresponding wild-type fragment with respect to their electrophoretic mobilities. These observations are indicative of different three-dimensional structures of these complexes which may reflect different forms of DNA bending induced in these otherwise identical fragments by complex formation with RNA polymerase.

Asunto(s)

RESUMEN

Escherichia coli lactose permease (also referred to as lactose carrier) is an integral protein of the cytoplasmic membrane. Using lactose permease either radiolabeled biosynthetically in plasmid-bearing E. coli minicells or radioalkylated post-synthetically by chemical modification, we have determined sites on the membrane-bound protein accessible to proteolytic attack and we have characterized several high-molecular-mass products. The most prominent polypeptide obtained from lactose permease radiolabeled biosynthetically is observed after digestion with different proteases. The fragment produced by thermolysin was shown to contain the intact N-terminus and to extend into the region around amino acid residue 140 which, according to secondary structure models, is presumed to be less tightly folded than the rest of the molecule. Evidence is presented that the corresponding fragments obtained after digestion with several other proteases also originate from the N-terminal part of the protein. This N-terminal segment of the lactose carrier is resistant to proteolytic digestion even in the presence of non-ionic detergents and it may represent a tightly folded domain. Additional proteolytic cleavage sites located C-terminal of the Cys148 residue can be inferred.

Asunto(s)

RESUMEN

Two operators are known to bind Escherichia coli galactose repressor with roughly equal affinity. A study of the control these two operators exert on the two overlapping gal promoters is reported. The experiments rest on a set of mutations specifically constructed to inactivate individual control units of the gal operon and on quantitation of gal promoter activities. Messenger RNAs initiated at one or other of the promoters in a cell-free transcription-translation system were determined by a primer extension assay with synthetic deoxyoligonucleotide primers. The main conclusions are: (i) the classical galactose operator O1, located upstream with respect to the two overlapping promoters is sufficient for negative control of the cAMP activated promoter P1; (ii) complete repression of the second promoter P2, on the other hand, needs the presence of both intact operators O1 and O2. Thus, the two overlapping gal promoters (with only 5 bp separating their respective transcriptional start sites) are both subject to negative control by the galactose repressor. This regulation, however, is exerted by two different mechanisms.

Asunto(s)

RESUMEN

The T-region of Ti-plasmids expresses four proteins (mol. wts. 74,000, 49,000, 28,000 and 27,000) in Escherichia coli minicells. Promoter activities are determined by sequences within the T-region, and the protein-coding regions map in that part of the T-region which is highly conserved in octopine and nopaline plasmids and which is responsible for shoot and root inhibition when expressed in plant cells. Three of the regions expressed in bacteria correlate with three regions which are transcribed in transformed plant cells; the fourth protein-coding region has no corresponding transcript in plants. At least three of the proteins synthesized in E. coli minicells are also expressed in cell-free systems prepared from E. coli and from Agrobacterium tumefaciens; the fourth protein (mol. wt. 49,000) is poorly expressed in both cell-free extracts. The possibility is discussed that the same genes are expressed in Agrobacteria and in transformed plant cells and that in both cases the gene products mediate growth regulatory effects to non-transformed plant cells.

Asunto(s)

RESUMEN

A series of plasmids has been constructed which contain either a single one of the two operators O1 and O2 of the Escherichia coli galactose operon or different combinations thereof. This permits comparison of the two operators with respect to their repressor binding ability. A plasmid containing only the second operator, O2, located within the structural gene galE, was found to titrate repressor in vivo and in vitro with essentially the same efficiency as a plasmid containing only the 'classical' galactose operator, O1, located upstream of the start of transcription. Whereas some cooperativity between the two sites seems possible in vivo, they bind repressor independently under the conditions of the in vitro assay. After hydroxylamine treatment of plasmid DNA in vitro, two different mutations have been isolated each of which inactivates the second operator, O2. Both are GC to AT transitions located at equivalent positions relative to the axis of rotational symmetry within the second gal operator. Subdividing O1 and O2 according to their 2-fold symmetry yields four half-sites with the consensus sequence (5')gTGnaAnC(3'). All known single point mutations of O1 and O2 affect the frame of invariant residues. The two half-sites of the lac operator also coincide with this frame.

Asunto(s)

RESUMEN

A protein of Mr 47,000 is synthesized in Escherichia coli minicells, when these harbor a multicopy plasmid carrying IS4 in either orientation and between different flanking sequences. The protein corresponds to the sequence predicted from the known DNA sequence of IS4, as shown by partial N-terminal radiolabel protein sequence analysis. Its apparent molecular weight, however, as determined from its electrophoretic mobility in SDS polyacrylamide gels, is smaller than predicted. When compared with other plasmid-encoded proteins, the IS4-encoded protein is synthesized in minicells in small amounts. Its synthesis has not been detected in a DNA-dependent cell-free system.

Asunto(s)

Asunto(s)

RESUMEN

The lacY gene product synthesised in vitro is identical to lactose permease isolated from cytoplasmic membranes as determined by apparent molecular weight and N-terminal amino acid sequence. The amino acid composition of the in vivo product agrees well with that predicted from the DNA sequence. The data assign the translational start on the DNA sequence and demonstrate that this protein is processed only by deformylation but not by proteolytic cleavage at the N-terminus.

Asunto(s)

RESUMEN

From a DNA-directed cell-free system, functional gal mRNA is obtained which directs the cell-free synthesis of the three galactose enzymes of Escherichia coli. A substantial fraction of this gal mRNA has the properties of a polycistronic messenger. Exposure to elevated temperatures in the presence or absence of magnesium ion results in pronounced changes of the capacity of this mRNA to give rise to the synthesis of the three enzymes. Depending on the conditions of the pre-treatment, the absolute amounts as well as the ratio of the three gene products synthesized can be changed. The different forms of gal messenger so obtained also exhibit different susceptibilities towards functional inactivation during the enzyme synthesis reaction. As the changes in template activity are reversible, it is concluded that the different treatments cause reversible transitions between different conformations of the gal mRNA.

Asunto(s)

Asunto(s)

RESUMEN

The strong repression of inducible synthesis of the enzymes of fatty acid degradation by glucose can be partially relieved by the addition of cyclic adenosine 3',5' monophosphate (cyclic AMP) to the growth medium. This reversal of the glucose effect by cyclic AMP is not observed in a mutant (K29) that is unable to grow on fatty acids as sole carbon source and that was found to synthesize low levels of several enzymes specified by the fad regulon. In a revertant selected for the ability to grow on oleate these effects are concomitantly relieved. By both genetic (co-transduction of the mutation with the strA locus) and biochemical experiments (an extract of the mutant strain does not show the cyclic AMP-dependent stimulation of the deoxyribonucleic acid-directed in vitro synthesis of the enzymes of the gal operon), it is demonstrated that the mutant lacks functional cyclic AMP receptor protein (CR protein). It is concluded that, like many other inducible enzyme systems, expression of the enzymes of the fad system requires cyclic AMP and the CR protein.

Asunto(s)

Asunto(s)

Asunto(s)

Asunto(s)