Martin Freundlich

The role of integration host factor In gene expression in Escherichia coli

Integration host factor is a sequence‐specific, histone‐like, multifunctional DNA‐binding and ‐bending protein of Escherichia coli. The characterization and functional analysis of this protein has been done mainly in bacteriophage λ and other mobile genetic elements. Less is known concerning the role of integration host factor (IHF) in E. coli, although it has been implicated in a number of processes in this organism including DNA replication, site‐specific recombination, and gene expression. This review presents recent work which suggests that IHF alters the activity of an unusually large number of operons in E. coli. We discuss the possible physiological relevance of the involvement of IHF in gene expression and the hypothesis that IHF is a member of a class of functionally redundant proteins that participate in chromosome structure and multiple processes involving DNA.

Expression of a valine-resistant acetolactate synthase activity mediated by the ilv O and ilv G genes of Escherichia coli K-12

SummaryA strain carrying the ilv0603 mutation has been isolated in E. coli K-12 and its characteristics were found to be very similar to those previously reported by Ramakrishnan and Adelberg (1965a) for other ilv0 mutants.The strain carrying the ilv0603 mutation is resistant to valine inhibition (Valr) and we show that this resistance depends on the expression of a newly recognized gene, ilvG, which is located at min 75, between ilvE and ilvD on the E. coli K-12 map. The ilvG gene causes the expression of a Valr acetolactate synthase, which is detectable only when the ilv0603 mutation is also present in cis on the same chromosome. Under these conditions the Valr acetolactate synthase activity is eluted, on a hydroxylapatite column, at an ionic strength slightly lower than that required for elution of the remaining acetolactate synthase activity (sensitive to valine inhibition). The Valr peak is missing in a strain carrying an ilvG (amber) mutation.

Detection of messenger RNA from the isoleucine-valine operons of Salmonella typhimurium by heterologous DNA-RNA hybridization: Involvement of transfer RNA in transcriptional repression

SummaryA hybridization assay using Escherichia coli K-12 DNA isolated from the specialized transducing bacteriophage λCI857St68h80 dilv was used to examine the rate of synthesis of the messenger RNAs (mRNA) derived from the isoleucine-valine (ilv) gene cluster of Salmonella typhimurium. In all cases examined, changes in ilv enzyme levels could be correlated with changes in the rate of synthesis of ilv mRNA. Several well characterized regulatory mutants of S. typhimurium had rates of synthesis of ilv mRNA 3 to 8-fold higher than the repressed wildtype strain. The increased rates of ilv mRNA synthesis found in a hisT strain as well as in isoleucyl-and leucyl-tRNA synthetase mutants, strongly suggests a role for branched-chain aminoacyl-tRNAs in transcriptional control.

Two forms of biosynthetic acetohydroxy acid synthetase in Salmonellatyphimurium

Summary The initial enzyme common to the isoleucine-valine biosynthetic pathway, acetohydroxy acid synthetase (AHAS), is inhibited by valine. Evidence is presented that there are two forms of this enzyme, one sensitive (AHASs) to inhibition by valine, and the other resistant (AHASr). AHASr is under multivalent repression by isoleucine, valine and leucine, while AHASs is repressed only by valine and leucine. Multiple forms of AHAS allow for the synthesis of isoleucine in the presence of excess valine.

In vitro interactions of integration host factor with the ompF promoter-regulatory region of Escherichia coli

SummaryPrevious work has shown that integration host factor (IHF) mutants have increased expression and altered osmoregulation of OmpF, a major Escherichia coli outer membrane protein. By in vitro analysis the possibility was investigated that IHF interacts directly with the ompF promoter region. Gel retardation assays and DNase I protection experiments showed that IHF binds to two sites in the ompF promoter region centered at positions −180 and −60 relative to the start of transcription. Gel electrophoresis studies with circularly permuted ompF promoter fragments indicated that IHF binding strongly increased a small intrinsic bend in the ompF promoter region. The addition of IHF to a purified in vitro transcription system strongly and specifically inhibited ompF transcription. This inhibition was reversed by increasing the concentration of OmpR, a positive activator required for ompF expression, suggesting that IHF may inhibit ompF transcription by altering how OmpR interacts with the ompF promoter.

Cloning and expression of theilvB gene ofEscherichia coli K-12

SummaryA plasmid containing theilvB operon, which codes for acetohydroxy acid synthase I ofEscherichia coli K-12, was isolated using a ligated mixture of DNA from plasmid pBR322 and FilvB4 treated with endonucleaseSalI. A shortened derivative of this plasmid was isolated by cloning a 3.4 kb bacterial fragment into plasmid pKEN005 to yield plasmid pTCN12. The orientation of theilvB operon relative to plasmid genes was determined by restriction enzyme mapping. Measurement of the level of the product of theilvB gene, acetohydroxy acid synthase I, indicated that plasmid pTCN12 contained a functionalilvB promoter and control region. The DNA from this plasmid was used as a probe to show that the rate of synthesis ofilvB mRNA was proportional to the levels of acetohydroxy acid synthase I.

Control of isoleucine, valine and leucine biosynthesis VII. Role of valine transfer RNA in repression

Abstract These studies support the hypothesis that charging of valine tRNA is a step required for repression, mediated by the valyl-tRNA synthetase. Consistent with this hypothesis were the observations that (a) dl - threo -α-amino-β-chlorobutyric acid (aminochlorobutyrate) which was activated and attached to valine tRNA blocked the synthesis of the isoleucine-valine biosynthetic enzymes, while dl -α-aminobutyric acid which was activated but not transferred to tRNA had no effect on the formation of these enzymes; (b) the effect of aminochlorobutyrate was due to repression and not to the production of false proteins, since the synthesis of numerous biosynthetic and inducible enzymes was not blocked by the analogue; (c) there was a direct correlation between the intracellular charging of valine tRNA by valine or aminochlorobutyrate and repression of the isoleucine-valine enzymes; (d) the steady-state charging of valine tRNA in vivo was virtually the same in a valine auxotroph grown with added valine or aminochlorobutyrate.

Integration host factor binds specifically to sites in the ilvGMEDA operon in Escherichia coli.

Integration host factor (IHF) of Escherichia coli is a histone-like protein that is involved both in site-specific recombination and in regulating the expression of a number of phage and bacterial genes. We have shown previously that transcription of the ilvGMEDA operon in E. coli is greatly reduced in IHF mutants. We report here that IHF specifically protects two sites within the ilvGMEDA promoter-regulatory region against DNase I digestion. These sites are located upstream from the promoter and in the leader region just prior to the sequence that specifies the attenuator. The footprinting experiments and gel retardation assays show that these sites have strong affinity for IHF. These data and results with ilvGMEDA-lac promoter fusions suggest a direct role for IHF in expression of the ilvGMEDA operon.

Transcriptional control of the isoleucine-valine messenger RNA's in E. coli K-12

SummaryHybridization of messenger ribonucleic acid (mRNA) isolated from Escherichia Coli K-12 to deoxyribonucleic acid (DNA) from λCI857st68h80dilv was used to detect isoleucine-valine (ilv) specific mRNA. A number of strains partially constitutive for the isoleucine-valine enzymes had levels of ilv mRNA 2 to 3-fold higher than the parent strain. Starvation for any of the branched-chain amino acids resulted in a 20 to 23-fold increase in ilv mRNA as compared to repressed levels. These differences were not due to altered growth rates or to changes in the stability of ilv mRNA. These data indicate that regulation of the isoleucine-valine enzymes by multivalent repression occurs mainly at the level of transcription. Kinetics of elongation of ilv mRNA after repression are consistent with the assumption that the mechanism of multivalent repression involves the prevention of further initiations by RNA polymerase.

Estimation of in vivo aminoacylation by periodate oxidation: tRNA alterations and iodate inhibition

Abstract Two problems associated with periodate oxidation in determining the extent of aminoacylation of tRNA are discussed. One of the products of this reaction, sodium iodate, was found to inhibit tRNA charging. In addition, periodate oxidation also appears to alter sites other than the 3′-end on at least two isoacceptor species of tRNA Leu .