Fine structure of the gradient of polarity in the z gene of the lac operon of Escherichia coli

110

The affinity of various mutant lac DNAs for the lac repressor was examined by means of a competitiod binding assay. The results indicate that there exist at least two binding sites for the lac repressor. The primary binding site is coincidental with the operator, as defined by genetic studies. Mutations of the promoter or the Z gene have little or no effect on the repressor binding properties of this primary binding site, thereby confirming that the operator is distinct from lacP and Z. The secondary binding site(s), which has a 25-to 31-fold lower affinity for the repressor, is-located within the operator proximal third of the Z gene.Associated with the lactose (lac) operon are a series of genetic signals that determine when and how often expression of the lac operon occurs. One of these signals is the promoter (P) where transcription initiation is thought to occur (1-4). Another signal is the operator (0), the target or binding site for the lac repressor (5-8). Genetic experiments have indicated that these signals are arranged in the following order, I-P-0-Z, where I and Z are the structural genes for the repressor and ,6-galactosidase, respectively, and that the operator is clearly separated from P and Z (9-11). The development of in vitro systems for studying repressor-operator interaction by Gilbert and Muller-Hill (7) and Riggs et al. (8) medium similar to TCL medium described by Riggs et al. (13); it was modified to contain 0.4 mg/100 ml of glucose instead of lactose and 0.54 mg/100 ml of casamino acids that had been treated to lower the phosphate contant (14)1.The buffers used include filter buffer (FB) and binding buffer (BB), both of which are similar to that described in Riggs et al. (13), except that the concentration of magnesium acetate was lowered to 2.5 mM. All phage dilutions were performed in X-Ca++ buffer (12).Genetic Markers of the lac Controlling Elements. The mutations used in these studies are described in Fig. 1 (15). The deletions after incorporation into Xptrp-lac phages were also mapped against the point mutations by infecting point mutant strains with the phage and looking for lac+ transductants. The lacZ point mutants have been described (16,17).Specialized Transducing Phages. Several of the mutations used in this study were incorporated into the 080plac (18) genome. The 480plac derivatives carrying the lac mutations S20, S146, P-L157, and P-L305 were isolated by a procedure described by Malamy et al. (19). The 080plac derivatives carrying the lacoO mutations, the double lac mutations L8-UV5, and the lacPrl9 mutation were isolated by growing the 480plac-S20 phage on the appropriate mutant strain and then plating the progeny with the X7062 indicator bacterium on T-XG plates. Blue (lac+) plaques were found at a frequency of from 10-3 to 10-4. Since S20 is a lacO-Z deletion, p1esumably lac+ phage can arise only by recombination events that transfer the desired oc mutation or P mutation onto the t,80plac genome at a high frequency.

mentioning

confidence: 99%

The Location of the Repressor Binding Sites in the lac Operon

Reznikoff

Winter

Hurley

1974

110

“…A second, independent approach makes use of reinitiation of translation after chain termination. Reinitiation at internal sites within cistrons was first described by Sarabhai and Brenner (5) in the rIIB cistron of phage T4, and later demonstrated by Zipser (6)(7)(8) and Newton (9,10) and their coworkers in the z gene of the lac operon of Escherichia coli. These studies showed that after chain termination at certain nonsense codons, reinitiation of polypeptide synthesis can occur.…”

mentioning

confidence: 99%

Reinitiation of a lac Repressor Fragment at a Codon Other Than AUG

Ganem

Miller

Files

et al. 1973

ABSTRACT52 Spontaneous nonsense mutants in the lac i gene of Escherichia coli were isolated and characterized. All mutants located early in the gene show negative complementation in vivo with a wild-type i gene in a recA diploid strain. In vitro studies show that those mutants that display negative complementing activity in vivo also make lac repressor fragments retaining inducer binding and immunological crossreactivity with wild-type repressor. Amino-acid sequence analysis of these fragments shows that they arise by reinitiation at internal sities of the i message after chain termination at a prior amber or ochre codon.There are at least two different internal reinitiation sites in the first 200 nucleotides of the translated part of the i message. The first site corresponds to the first internal in phase AUG codon, which specifies the methionine residue at position 42 of the repressor protein. This site can be activated by an amber codon, 45 nucleotides before the AUG codon. The second site is only 60 nucleotides past the first site and can be activated by an amber mutation derived from residue 60 of the protein. The second initiation codon specifies the amino-acid leucine in the wild-type repressor, but the reinitiated fragment shows an aminoterminal methionine residue at this position. Therefore, the second initiation site seems to involve an in vivo ambiguity of the genetic code in that the same codon can be translated into two different amino acids depending on the recognition of this codon during initiation (when methionine is inserted) or elongation of protein synthesis (when leucine is inserted). The possibility that a codon other than AUG can act as an initiation codon in vivo is discussed.One object of our current work is to understand how a cell directs the initiation of translation of messenger RNA (mRNA) to occur at specific nucleotide sequences and also to determine the exact nature of these sequences. One approach to this problem has been the direct isolation and sequence analysis of ribosomal initiation sites (1-4). A second, independent approach makes use of reinitiation of translation after chain termination. Reinitiation at internal sites within cistrons was first described by Sarabhai and Brenner (5) in the rIIB cistron of phage T4, and later demonstrated by and Newton (9, 10) and their coworkers in the z gene of the lac operon of Escherichia coli. These studies showed that after chain termination at certain nonsense codons, reinitiation of polypeptide synthesis can occur.Recently, we have shown that an early spontaneous amber mutation in the i gene gives rise to translational reinitiation; reinitiation occurs past the amber block at the first internal AUG codon of the repressor mRNA (11,12

“…Newton (4,5), and Zipser and others (6)(7)(8) have found initiation sites within the z-gene of the lac operon in Escherichia coli. These have no effect during normal translation, and do not affect the gene product, jB-galactosidase.…”

mentioning

confidence: 99%

Translational Restarts: AUG Reinitiation of a lac Repressor Fragment

Platt

Weber

Ganem

et al. 1972

An early, spontaneous amber mutation in the lac i-gene allows translational reinitiation, which results in a mutant lac repressor. Comparison of the amino-terminal sequence of this mutant repressor with the partial amino-acid sequence of the wild-type lac repressor shows that reinitiation occurs at the first internal AUG codon, and results in a mutant protein lacking 42 residues at the amino-terminal end. This protein binds the inducer isopropyl-fl-D-thiogalactoside with normal affinity, and is capable of maintaining a tetrameric structure; however, it does not repress in vivo. These data suggest that the amino-terminal portion of the wildtype lac repressor is necessary either for direct binding to the lac operator or for the correct conformation for binding to DNA.The codon AUG functions as an initiation signal in protein biosynthesis (1). An AUG triplet occurring internally codes for the insertion of methionine; under normal conditions, no initiation occurs within a gene. However, internal initiation sites have been found in certain cistrons. These sites have little or no effect on the wild-type gene product, and function only in the presence of a nearby terminator codon, permitting the reinitiation of translation and the synthesis of a C-terminal fragment of the gene product. Once the site is made available by chain termination, initiation presumably occurs by the normal mechanism (2). Sarabhai and Brenner have described a reinitiation site in the rII-B cistron of bacteriophage T4; chain termination is required for this site to function in initiation (3). Both the nonsense fragment and a C-terminal fragment of the rII-B gene product are synthesized, and the insertion of frameshift mutations shows that reinitiation is independent of the phase between the nonsense codon and the starter site.Newton (4, 5), and Zipser and others (6-8) have found initiation sites within the z-gene of the lac operon in Escherichia coli. These have no effect during normal translation, and do not affect the gene product, jB-galactosidase. As in the rII-B cistron, however, chain termination close to one of these sites results in the synthesis of a reinitiated polypeptide, which is the C-terminal fragment of fl-galactosidase, that can be detected both in vivo and in vitro (8,9).These results demonstrate that internal initiation signals exist, and are able to function in protein biosynthesis under certain conditions. It is not known what information is necessary for initiation beyond the requirement for an initiating codon.In a genetic study to be described in a subsequent paper, we have demonstrated the existence of two natural reinitiation sites early in the i-gene of the lac operon of E. coli. We report here the characterization of a lac repressor polypeptide that arises by reinitiation at the earlier of these sites as the result of chain termination at an amber codon. To locate the site of reinitiation, we determined the sequence of the amino-terminal regions of the mutant and wild-type repressors. Our results imply that rei...