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R Landick

Publications and source records attributed to R Landick.

69 records · Page 4Linked to original sources
PubMed1986

Rho-dependent transcription termination in the tryptophanase operon leader region of Escherichia coli K-12.

Recent studies have suggested that expression of the tryptophanase (tna) operon of Escherichia coli is subject to transcription termination-antitermination control (V. Stewart and C. Yanofsky, J. Bacteriol. 164:731-740, 1985). In vivo studies have indicated that the transcribed leader region, tnaL, contains a site or sites of rho-dependent transcription termination (rho is the polypeptide product of the gene rho). We now report direct in vitro evidence that tnaL contains rho-dependent termination sites. In vivo termination appeared to occur at the rho-dependent termination sites identified in vitro. Transcription pausing analyses correlated sites of pausing in tnaL with sites of rho-dependent termination.

Base Sequence↗
PubMed1986

Cloning and characterization of livH, the structural gene encoding a component of the leucine transport system in Escherichia coli.

The physical location of the genetically defined livH gene was mapped in the 17-kilobase plasmid pOX1 by using transposon Tn5 inactivation mapping and further confirmed by subcloning and complementation analysis. These results indicated that the livH gene maps 3' to livK, the gene encoding the leucine-specific binding protein. Moreover, the nucleotide sequence of the livH gene and its flanking regions was determined. The livH gene is encoded starting 47 base pairs downstream from the livK gene, and it is transcribed in the same direction as the livK gene. The livK-livH intergenic region lacks promoter sequences and contains a GC-rich sequence that could lead to the formation of a stable stem loop structure. The coding sequence of the livH gene, which is 924 base pairs, specifies a very hydrophobic protein of 308 amino acid residues. Expression of livH-containing plasmids in minicells suggested that a poorly expressed protein with an Mr of 30,000 could be the livH gene product.

Amino Acid Sequence↗
PubMed1985

The complete nucleotide sequences of the Escherichia coli LIV-BP and LS-BP genes. Implications for the mechanism of high-affinity branched-chain amino acid transport.

The Escherichia coli LIV-I and LS amino acid transport systems are high-affinity, periplasmic, binding protein-dependent systems that utilize the leucine-, isoleucine-, valine-binding protein (LIV-BP) and leucine-specific binding protein (LS-BP), respectively. These two binding proteins (BPs) interact with a common set of membrane proteins to transport branched-chain amino acids into the cytoplasm. The two BP genes are encoded in a regulon at minute 76 of the E. coli chromosome that also contains the genes for the common membrane protein components. We report here the nucleotide and deduced amino acid sequences for the LIV-BP and LS-BP genes and their protein products. Both BPs are encoded with a 23-amino acid signal sequence that is removed when the BPs are secreted into the periplasm. We have examined the pattern of amino acid sequence conservation between the two BP molecules and, by comparison of the predicted secondary structures to the 2.0-A crystal structure of the LIV-BP, have found regions of the BPs that may be involved in membrane protein interaction. We also analyzed the translational efficiency of the two BP mRNAs as determined by their nucleotide sequences.

Amino Acid Sequence↗
PubMed1985

The leucine binding proteins of Escherichia coli as models for studying the relationships between protein structure and function.

The genes encoding the leucine binding proteins in E coli have been cloned and their DNA sequences have been determined. One of the binding proteins (LIV-BP) binds leucine, isoleucine, valine, threonine, and alanine, whereas the other (LS-BP) binds only the D- and L-isomers of leucine. These proteins bind their solutes as they enter the periplasm, then interact with three membrane components, livH, livG, and livM, to achieve the translocation of the solute across the bacterial cell membrane. Another feature of the binding proteins is that they must be secreted into the periplasmic space where they carry out their function. The amino acid sequence of the two binding proteins is 80% homologous, indicating that they are the products of an ancestral gene duplication. Because of these characteristics of the leucine binding proteins, we are using them as models for studying the relationships between protein structure and function.

Amino Acid Sequence↗
PubMed1985

Translation activates the paused transcription complex and restores transcription of the trp operon leader region.

It has been proposed that RNA polymerase pausing in the leader region of the tryptophan (trp) operon of Escherichia coli is responsible for the synchronization of transcription and translation essential to attenuation control. In this report we use an in vitro coupled transcription/translation system to study the effect of trp leader peptide synthesis on RNA polymerase pausing in the trp leader region. Wild-type and translation-defective trp leader templates of E. coli and Serratia marcescens were employed, and pause RNA synthesis and paused complex release (activation) were quantified relative to synthesis of the terminated leader transcript. It was observed that pausing in the trp leader region was prolonged when translation of the leader transcript was reduced by mutations in the leader region or by addition of the translation inhibitor kasugamycin or chloramphenicol. Experiments with S-30 extracts from a mutant strain that is inefficient in translating the tryptophan codons in the leader transcript indicated that ribosome movement to these codons also releases the paused transcription complex. These findings indicate that the paused trp leader transcription complex resumes transcription when released by ribosome movement over the leader peptide coding region. This release would facilitate the coupling of transcription and translation essential to attenuation control.

Amino Acid Sequence↗
PubMed1984

Stability of an RNA secondary structure affects in vitro transcription pausing in the trp operon leader region.

Transcription of the tryptophan (trp) operon of Escherichia coli and other bacterial species is regulated by the formation of alternative secondary structures in the leader segment of the transcript. During in vitro transcription of the trp leader region RNA polymerase pauses at base pair 92 after synthesis of an RNA hairpin secondary structure. We studied the dependence of pausing on hairpin stability by examining mutant trp templates containing base pair substitutions in the region corresponding to the hairpin secondary structure. Base changes that lower the stability of the hairpin were found to reduce both the frequency and half-life of RNA polymerase pausing while base changes that do not affect hairpin stability had little effect on pausing. Pausing was enhanced by the nusA protein; this enhancement was greatly reduced on mutant templates specifying less stable hairpins. The frequency of pausing on some mutant templates was correlated with the extent of read-through transcription beyond the trp attenuator, suggesting a possible role for pausing in the coupling of transcription and translation during transcription of the leader region of the operon.

DNA-Directed RNA Polymerases↗
PubMed1984

Secretion and degradation of mutant leucine-specific binding protein molecules containing C-terminal deletions.

The leucine-specific binding protein (LS-BP), a periplasmic component of the Escherichia coli high-affinity leucine transport system, is initially synthesized in a precursor form with a 23 amino acid N-terminal leader sequence that is removed during secretion of the protein into the periplasm. Using in vitro mutagenesis, deletion mutants of the LS-BP gene have been constructed with altered or missing amino acid sequences in the C-terminal portion of the protein. These altered binding proteins exhibited normal processing and secretion but were rapidly degraded in the periplasmic space. In the presence of an uncoupler of the transmembrane potential (CCCP) the precursor forms accumulated in the membrane and were protected from degradation. The altered binding proteins also were secreted by spheroplasts of E coli, after which they were easily detected.

Bacterial Proteins↗
PubMed1984

Role of membrane potential in protein folding and domain formation during secretion in Escherichia coli.

The synthesis and processing of the periplasmic components of the leucine transport system of E coli have been studied to determine the role played by transmembrane potential in protein secretion. Both the leucine-isoleucine-valine binding protein and the leucine-specific binding protein are synthesized as precursors with 23 amino acid N-terminal leader sequences. The processing of these precursors is sensitive to the transmembrane potential. Since the amino acid sequence and the crystal structure have been determined for the leucine-isoleucine-valine binding protein, it and the closely related leucine-specific binding protein represent convenient models in which to examine the mechanism of protein secretion in E coli. A model for secretion has been proposed, suggesting a role for transmembrane potential. In this model, the N-terminal amino acid sequence of the precursor is assumed to form a hairpin of two helices. The membrane potential may orient this structure to make it accessible to processing. In addition, the model suggests that a negatively charged, folded domain of the secretory protein may electrophorese toward the trans-positive side of the membrane, thus providing an additional role for the transmembrane potential.

Bacterial Proteins↗
PubMed1984

Nucleotide sequence of the heat shock regulatory gene of E. coli suggests its protein product may be a transcription factor.

We have sequenced a cloned segment of E. coli chromosomal DNA that includes the heat shock regulatory gene htpR. This segment contains an 852 nucleotide open reading frame bounded by transcriptional and translational signals. Both in vivo and in vitro the cloned segment produces a single protein that migrates in gels with the cellular protein (F33.4) implicated as the htpR product. Properties of a cloned fragment of the coding sequence truncated at the promoter-distal end are consistent with this assignment. The htpR gene product appears homologous to the sigma factor of RNA polymerase, and the two proteins are predicted to have similar secondary structure. In addition, two regions of the predicted htpR product resemble protein-DNA contact points conserved in known DNA-binding proteins.

Amino Acid Sequence↗
PubMed1984

Optimization of polyacrylamide gel electrophoresis conditions used for sequencing mixed oligodeoxyribonucleotides.

We investigated two components of the polyacrylamide gel electrophoresis system used for sequencing DNA to improve the system for sequencing synthesized oligodeoxyribonucleotides containing positions of degeneracy. First, we varied the ratio of methylene-bis-acrylamide (MBA) to acrylamide from that commonly used in DNA sequencing gels (1% MBA:19% acrylamide). A moderate increase in the MBA:acrylamide ratio proves optimal when sequencing is used to confirm that a synthesized fragment contains equivalent stoichiometries of the different nucleotides at a given position of degeneracy. Such information is particularly important if the degenerate oligodeoxyribonucleotide preparation is to be employed as a hybridization probe. A further increase in the MBA:acrylamide ratio (3% MBA:19% acrylamide) produces a gel in which the sequence of an oligodeoxyribonucleotide mixture containing several positions of degeneracy can be read most easily. Increasing the MBA acrylamide ratio suppresses the effect of base composition on electrophoretic mobility of a fragment. Second, we investigated the use of a Tris-citrate buffer system in place of the standard Tris-borate system. We found the Tris-citrate system to be significantly more effective in preventing discontinuities in the banding pattern of the smaller fragments. Finally, we show that high MBA gels are also effective in resolving mixtures of oligoribonucleotides such as those produced by T1 ribonuclease digestion of small RNAs.

Base Sequence↗
PubMed1980

The in vitro synthesis and processing of the branched-chain amino acid binding proteins.

The synthesis of the leucine-specific and LIV-binding proteins was examined in vitro in a coupled transcription/translation system using the hybrid plasmids pOX7 and pOX13 as templates. Plasmid pOX7 contains the livK gene coding for the leucine-specific binding protein and pOX13 contains the livJ gene coding for the LIV-binding protein. Both binding proteins were synthesized in vitro as precursor forms with molecular weights approximately 2,500 greater than their respective mature forms. Conversion of the precursor forms to their mature forms occurred during post-translational incubation following synthesis in the presence of membrane. The precursor of the LIV-binding protein was processed more rapidly than the leucine-specific binding protein precursor. Processing activity could be removed from the in vitro synthesis system by centrifugation, suggesting that the processing activity was membrane associated. Restoration of post-translational processing activity was achieved by adding inside-out membrane vesicles to membrane-depleted reaction mixtures.

Amino Acids, Branched-Chain↗
PubMed1980

Regulation of high-affinity leucine transport in Escherichia coli.

Leucine is transported into E coli by two osmotic shock-sensitive, high-affinity systems (LIV-I and leucine-specific systems) and one membrane bound, low-affinity system (LIV-II). Expression of the high-affinity transport systems is altered by mutations in livR and 1stR, genes for negatively acting regulatory elements, and by mutations in rho, the gene for transcription termination. All four genes for high-affinity leucine transport (livJ, livK, livH, and livG) are closely linked and have been cloned on a plasmid vector, pOX1. Several subcloned fragments of this plasmid have been prepared and used in complementation and regulation studies. The results of these studies suggest that livJ and livK are separated by approximately one kilobase and give a gene order of livJ-livK-livH. livJ and livK appear to be regulated in an interdependent fashion; livK is expressed maximally when the livJ gene is activated by mutation or deletion. The results support the existence of separate promotors for the livJ and livK genes. The effects of mutations in the rho and livR genes are additive on one another and therefore appear to be involved in independent regulatory mechanisms. Mutations in the rho gene affect both the LIV-I and leucine-specific transport systems by increasing the expression of livJ and livK, genes for the LIV-specific and leucine-specific binding proteins, respectively.

Biological Transport↗
PubMed1980

Structural and functional analysis of cloned DNA containing genes responsible for branched-chain amino acid transport in Escherichia coli.

The four genes encoding the components of the high-affinity branched-chain amino acid transport systems in Escherichia coli (livH, livG, livJ, and livK) have been cloned into lambda phage and subsequently into the plasmid vector pACYC184. The presence of the four structural genes and their accompanying regulatory regions on the resultant plasmid, pOXI, was confirmed by genetic complementation and analysis and by transport studies carried out on the appropriate transformed mutant strains. When pOX1 DNA was used to direct an in vitro transcription/translation system, four major polypeptide products were produced. Immunoprecipitation with antibody directed against the LIV-binding protein identified the two leucine-binding proteins as products of in vitro synthesis. The binding proteins were produced in precursor forms and had molecular weights approximately 2500 higher than the processed, mature forms. A minicell-producing strain transformed with plasmid pOX1 produced the binding proteins in the processed form.

Amino Acids, Branched-Chain↗
PubMed1980

Amino-terminal sequence and processing of the precursor of the leucine-specific binding protein, and evidence for conformational differences between the precursor and the mature form.

A 2.1-kilobase Bgl II DNA fragment from Escherichia coli containing livK, the gene coding for the leucine-specific binding protein, has been cloned into the BamHI site of the plasmid vector pBR322. The DNA sequence of segments of the resulting plasmid, pOX7, established the location of the livK gene and the direction of its transcription. In vitro protein synthesis directed by pOX7DNA yielded the Mr 42,000 precursor of the leucine-specific binding protein and a small amount of the Mr 39,000 mature protein. Continued incubation of the in vitro reaction mixture after DNase and RNase treatment resulted in additional processing. The DNA sequence of the beginning of livK suggested that 23 additional amino acid residues are present as an extension of the NH2 terminus of the mature protein. Amino acid sequence analysis established that the precursor has the predicted 23-residue extension. Proteolytic digestion studies with the precursor and mature forms of the leucine-specific binding protein indicate that there are conformational differences between the two. This suggests a possible role for the signal sequence in determining the conformation of the binding protein precursor that is recognized by the membrane.

Amino Acid Sequence↗