Geoffrey Rowland - Academia.edu (original) (raw)

Papers by Geoffrey Rowland

Research paper thumbnail of Mapping of trypsin cleavage and antibody-binding sites and delineation of a dispensable domain in the beta subunit of Escherichia coli RNA polymerase

Journal of Biological Chemistry, Dec 1, 1991

We have mapped principal sites in the Escherichia coli RNA polymerase molecule that are exposed t... more We have mapped principal sites in the Escherichia coli RNA polymerase molecule that are exposed to attack by trypsin under limited proteolysis conditions. The 1342-amino acid-long fi subunit is alternatively cleaved at Argeo3 or Lyseos. The cleavage occurs adjacer,t to a dispensable domain (residues 940-1040) that is absent in the homologous RNA polymerase subunits from chloroplasts, eukaryotes, and archaebacteria. In E. coli, this region can be disrupted with genetic deletions and insertions without the loss of RNA polymerase function. Insertion of 127 amino acids into this region introduces a new highly labile site for trypsin proteolysis. The dispensable domain carries the epitope for monoclonal antibody PYN-6 (near residue lOOO), which can be used for anchoring the catalytically active enzyme on a solid support. We also report the identification of a secondary trypsin cleavage at Ar8' of the B' subunit within a putative zinc-binding domain that is conserved in prokaryotes and chloroplasts. DNA-dependent RNA polymerase plays a central role in the expression and regulation of genes. The best characterized RNA polymerase is the enzyme from Escherichia coli, which can carry out in uitro all basic biochemical reactions of the transcription cycle (reviewed in Refs. 1-3). The RNA polymerase molecule also accepts a myriad of regulatory signals that modulate the basic reactions (4). The multiplicity of RNA polymerase function is reflected in its structural complexity. The E. coli holoenzyme (a&?'(r) is composed of the catalytic core component (a2pP') and the loosely associated initiation factor u7', which is replaceable by other u factors with different promoter specificities (5). The core subunits are composed of 1407 (p'), 1342 (p), and 329 (a) amino acid residues (6) and carry no function individually. They can be separated * This work was supported in part by American Cancer Society Grant MV-285

Research paper thumbnail of Cloning and physical mapping of the Staphylococcus aureus rplL, rpoB and rpoC genes, encoding ribosomal protein L7/L12 and RNA polymerase subunits   and  

Journal of General Microbiology, 1992

Using segments of the Escherichia coli rpoB and rpoC genes as heterologous probes, we have identi... more Using segments of the Escherichia coli rpoB and rpoC genes as heterologous probes, we have identified and cloned an 8.3 kb PstI fragment from the Staphylococcus aureus genome containing the rpoB and rpoC genes, which respectively encode the p and p' subunits of DNA-directed RNA polymerase. This region is almost certainly equivalent to the riflocus, located near tofus at interval 12/13 on the S. aureus linkage map. Limited DNA sequencing revealed the gene order rpoB-rpoC (transcribed from left to right) and identified the rplL gene, encoding ribosomal protein L7/L12, upstream of rpoB. This and other evidence suggests that the rpoBC genes of S. aureus form part of a large gene cluster encoding components of the transcription and translation apparatus which is well-conserved in other eubacteria.

Research paper thumbnail of In vivo cloning of a carboxy-terminal rpoB allele which confers altered transcriptional properties

Folia Microbiologica, 1995

... would like to thank K. llammer and N. Fill for providing the plasmid pNF1931, F. Corpct for t... more ... would like to thank K. llammer and N. Fill for providing the plasmid pNF1931, F. Corpct for the program Mul-talin, and Maggie Smith and ... BORUIOlOV S., SEVERINOV K., KASiIE.EV M., LEBEDEV A., BASS I., ROWLAND GC, L,M P.-P., GLASS RE, NIKIFOROV V., GOLDFARB A ...

Research paper thumbnail of Nucleotide and deduced amino acid sequence of the gene for a novel protein with a possible regulatory function encoded in the β operon of Staphylococcus aureus

FEMS Microbiology Letters, 1995

Although considerable homology exists between the translation products of the rpL?+ rpoB and rpoC... more Although considerable homology exists between the translation products of the rpL?+ rpoB and rpoC genes of the p operons of the Gram-negative organism Escherichia coli and the Gram-positive Staphylococcus aureus the region between the rplL and rpoB genes is quite different in the two bacterial species. In E. coli the 324 bp has three centres of dyad symmetry in the first half of the sequence and multiple nonsense codons in all three reading frames. By contrast, the corresponding region in S. aureur consists of 1000 bp capable of forming a similar arrangement of stem-loop structures but with an open reading frame, sited 177 bp downstream of the end of rplL and 217 bp upstream of the beginning of the rpoB gene, with consensus initiation and termination signals, which if translated would generate a 22,665 Da protein with 202 amino acids. In view of the inability to find any significant homology with other proteins in the data bank and because the evidence suggests, as in E. coli, that the rpLrpoB intergenic sequence is involved in regulation it is proposed that the expression product of orf202 may be a further element of control in the S. aureus /I operon.

Research paper thumbnail of What The Papers Say: Conservation of RNA polymerase

Research paper thumbnail of Nucleotide sequence of the Staphylococcus aureus RNA polymerase rpoB gene and comparison of its predicted amino acid sequence with those of other bacteria

Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1995

Tn5405 is a 12 kb staphylococcal composite transposon delimited by two inverted copies of the ins... more Tn5405 is a 12 kb staphylococcal composite transposon delimited by two inverted copies of the insertion sequence IS1 182. This transposon carries two aminoglycosides resistance genes, aphA-3 and aadE, an altered gene similar to sat4 from Campylobacter coli BElG4, and three open reading frames of unknown functions.

Research paper thumbnail of Phenotypic properties of a unique rpoA mutation (phs) of Escherichia coli

Journal of Bacteriology, 1985

The phs mutation of Escherichia coli has been suggested to affect the Na+/H+ antiport (D. Zilbers... more The phs mutation of Escherichia coli has been suggested to affect the Na+/H+ antiport (D. Zilberstein, E. Padan, and S. Schuldiner, FEBS Lett. 168:327-330, 1980). We have recently shown that the mutation affects the rpoA gene and thus affects transcription. The extent of the pleiotropy of the phs mutation was investigated. In addition to the previously reported growth defect on L-glutamate and melibiose, the mutation also affects at least two other metabolic systems. The transport and metabolism of arabinose is impaired and the transport of sulfate is reduced. The extent to which the effects of the phs mutation on metabolism are due to a defect in the Na+/H+ antiport was investigated, and no causal role for this transport system in the metabolic defects was found.

Research paper thumbnail of Genetic studies of the phs locus of Escherichia coli, a mutation causing pleiotropic lesions in metabolism and pH homeostasis

FEBS Letters, 1984

ft is well established that a wide range of bacteria exhibit a remarkable degree of cytoplasmic p... more ft is well established that a wide range of bacteria exhibit a remarkable degree of cytoplasmic pH bomeost~sis despite vexatious in external pH [t,2], However, the molecular m~b~isms which contribute to this ~omeostasis are only vaguely understood [Z]. The development of our understand~ug of many regulatory phenomena in bacteria has been ~o~seq~e~t upon genetic studies [3-s]. Thus, the isoiation of mut~ts defective in pH hom~st~is has been a major aim of workers in the field [6-S]. Of such mutants one of the most im~rtant has been a m~tatjon described as aff~ti~~ the Na'fH" antiport in E~~~~~c~~~ coli f@s) which results in a failure to grow at aika~~ne pH [&-X0], The p&s mutant was isolated after UV rn~~ge~~~s and ~~~~~~~ e~ri~hrn~nf as a stray unable to grow on either meiib~ose or glutamate ]SJ. Both of these carbon sources are believed to be taken up predominantly by Na~~dep~ndent meshes. These systems are dependent upon an Na* gradient ~tabl~shed by the Na*lH" antipo~ (11 3 1 Z]. Loss of the Na*/N" antiport would be ex

Research paper thumbnail of phs Locus of Escherichia coli, a mutation causing pleiotropic lesions in metabolism, is an rpoA allele

Journal of bacteriology, 1985

The phs mutation, which causes a pleiotropic growth defect, has been mapped and shown to be an al... more The phs mutation, which causes a pleiotropic growth defect, has been mapped and shown to be an allele of rpoA, the gene for the alpha subunit of RNA polymerase. The mutation is shown to cause a transcription defect in the arabinose operon, araBAD.

Research paper thumbnail of Refined structure-function analysis of the β-subunit of Escherichia coli RNA polymerase

Biochemical Society Transactions, 1989

Research paper thumbnail of Roles of the trkB and trkC gene products of Escherichia coli in K+ transport

Biochimie, Jan 31, 1985

Summary N Mutations at the trkB andtrkC loci ofEscherichia coli produce an abnormal efflux of K +... more Summary N Mutations at the trkB andtrkC loci ofEscherichia coli produce an abnormal efflux of K +. Tile mutations are partially dominant in diploids and revert frequently by what appears to be intragenic suppression to the null state. Tile mutations can be reverted by insertion of TnlO hlto the mutated gene, and spontaneous revertants are fidly recessive to the mutant allele hi diploids. K* efflux produced by NEM* and by DNP* persists h~ strahls with presumed null mutations, at either locus, hldicathlg neither gene product is the primary target for the effect of these hlhibitors on K* efflux.

Research paper thumbnail of Site-directed mutagenesis of the aromatic amino acid aminotransferase of Escherichia coli

Biochemical Society Transactions, 1989

Research paper thumbnail of d-Alanine transport in Escherichia coli

Biochemical Society Transactions, 1984

Research paper thumbnail of Comparative sequence analysis and predicted phylogeny of the DNA-dependent RNA polymerase beta subunits of Staphylococcus aureus and other eubacteria

Biochemical Society transactions, 1993

Research paper thumbnail of Antigenicity of the Aeromonas salmonicida maltose-inducible outer membrane porin (maltoporin) and a hybrid protein derived from it

Biochemical Society transactions, 1993

Research paper thumbnail of Properties and physiology of energy-coupled mutants of Escherichia coli

Biochemical Society transactions, 1984

Research paper thumbnail of ‘Stop-codon-specific’ restriction endonucleases: their use in mapping and gene manipulation

Gene, 1992

Certain restriction endonucleases recognise target sequences that contain the stop triplet TAG an... more Certain restriction endonucleases recognise target sequences that contain the stop triplet TAG and are commonly either 4 or 6 bp in length. Interestingly, these restriction targets do not occur at the frequency expected on the basis of base composition and size. For example, the tetranucleotide MaeI recognition sequence (CTAG) occurs considerably less commonly (5-8-fold) in the genome of Escherichia coli (and many other eubacteria) than expected from mononucleotide frequencies. This surprising rarity is particularly evident in protein-encoding genes and is largely dictated by codon usage. Thus, amber (TAG) nonsense mutations frequently give rise to novel MaeI (CTAG) sites which are unique within a translated region. Such amber/MaeI sites, whether arising spontaneously or created in vitro by site-directed mutagenesis, act as a useful physical marker for the presence of the nonsense mutation and are a convenient startpoint for a range of diverse procedures. These features provide a useful supplement to protein engineering methods which use nonsense suppression to mediate amino acid replacements.

Research paper thumbnail of Roles of the trkB and trkC gene products of Escherichia coli in K+ transport

Biochimie, 1985

Summary N Mutations at the trkB andtrkC loci ofEscherichia coli produce an abnormal efflux of K +... more Summary N Mutations at the trkB andtrkC loci ofEscherichia coli produce an abnormal efflux of K +. Tile mutations are partially dominant in diploids and revert frequently by what appears to be intragenic suppression to the null state. Tile mutations can be reverted by insertion of TnlO hlto the mutated gene, and spontaneous revertants are fidly recessive to the mutant allele hi diploids. K* efflux produced by NEM* and by DNP* persists h~ strahls with presumed null mutations, at either locus, hldicathlg neither gene product is the primary target for the effect of these hlhibitors on K* efflux.

Research paper thumbnail of What The Papers Say: Conservation of RNA polymerase

Research paper thumbnail of phs Locus of Escherichia coli, a Mutation Causing Pleiotropic Lesions in Metabolism, Is an rpoA Allele

The phs mutation, which causes a pleiotropic growth defect, has been mapped and shown to be an al... more The phs mutation, which causes a pleiotropic growth defect, has been mapped and shown to be an allele of rpoA, the gene for the a subunit of RNA polymerase. The mutation is shown to cause a transcription defect in the arabinose operon, araBAD.

Research paper thumbnail of Mapping of trypsin cleavage and antibody-binding sites and delineation of a dispensable domain in the beta subunit of Escherichia coli RNA polymerase

Journal of Biological Chemistry, Dec 1, 1991

We have mapped principal sites in the Escherichia coli RNA polymerase molecule that are exposed t... more We have mapped principal sites in the Escherichia coli RNA polymerase molecule that are exposed to attack by trypsin under limited proteolysis conditions. The 1342-amino acid-long fi subunit is alternatively cleaved at Argeo3 or Lyseos. The cleavage occurs adjacer,t to a dispensable domain (residues 940-1040) that is absent in the homologous RNA polymerase subunits from chloroplasts, eukaryotes, and archaebacteria. In E. coli, this region can be disrupted with genetic deletions and insertions without the loss of RNA polymerase function. Insertion of 127 amino acids into this region introduces a new highly labile site for trypsin proteolysis. The dispensable domain carries the epitope for monoclonal antibody PYN-6 (near residue lOOO), which can be used for anchoring the catalytically active enzyme on a solid support. We also report the identification of a secondary trypsin cleavage at Ar8' of the B' subunit within a putative zinc-binding domain that is conserved in prokaryotes and chloroplasts. DNA-dependent RNA polymerase plays a central role in the expression and regulation of genes. The best characterized RNA polymerase is the enzyme from Escherichia coli, which can carry out in uitro all basic biochemical reactions of the transcription cycle (reviewed in Refs. 1-3). The RNA polymerase molecule also accepts a myriad of regulatory signals that modulate the basic reactions (4). The multiplicity of RNA polymerase function is reflected in its structural complexity. The E. coli holoenzyme (a&?'(r) is composed of the catalytic core component (a2pP') and the loosely associated initiation factor u7', which is replaceable by other u factors with different promoter specificities (5). The core subunits are composed of 1407 (p'), 1342 (p), and 329 (a) amino acid residues (6) and carry no function individually. They can be separated * This work was supported in part by American Cancer Society Grant MV-285

Research paper thumbnail of Cloning and physical mapping of the Staphylococcus aureus rplL, rpoB and rpoC genes, encoding ribosomal protein L7/L12 and RNA polymerase subunits   and  

Journal of General Microbiology, 1992

Using segments of the Escherichia coli rpoB and rpoC genes as heterologous probes, we have identi... more Using segments of the Escherichia coli rpoB and rpoC genes as heterologous probes, we have identified and cloned an 8.3 kb PstI fragment from the Staphylococcus aureus genome containing the rpoB and rpoC genes, which respectively encode the p and p' subunits of DNA-directed RNA polymerase. This region is almost certainly equivalent to the riflocus, located near tofus at interval 12/13 on the S. aureus linkage map. Limited DNA sequencing revealed the gene order rpoB-rpoC (transcribed from left to right) and identified the rplL gene, encoding ribosomal protein L7/L12, upstream of rpoB. This and other evidence suggests that the rpoBC genes of S. aureus form part of a large gene cluster encoding components of the transcription and translation apparatus which is well-conserved in other eubacteria.

Research paper thumbnail of In vivo cloning of a carboxy-terminal rpoB allele which confers altered transcriptional properties

Folia Microbiologica, 1995

... would like to thank K. llammer and N. Fill for providing the plasmid pNF1931, F. Corpct for t... more ... would like to thank K. llammer and N. Fill for providing the plasmid pNF1931, F. Corpct for the program Mul-talin, and Maggie Smith and ... BORUIOlOV S., SEVERINOV K., KASiIE.EV M., LEBEDEV A., BASS I., ROWLAND GC, L,M P.-P., GLASS RE, NIKIFOROV V., GOLDFARB A ...

Research paper thumbnail of Nucleotide and deduced amino acid sequence of the gene for a novel protein with a possible regulatory function encoded in the β operon of Staphylococcus aureus

FEMS Microbiology Letters, 1995

Although considerable homology exists between the translation products of the rpL?+ rpoB and rpoC... more Although considerable homology exists between the translation products of the rpL?+ rpoB and rpoC genes of the p operons of the Gram-negative organism Escherichia coli and the Gram-positive Staphylococcus aureus the region between the rplL and rpoB genes is quite different in the two bacterial species. In E. coli the 324 bp has three centres of dyad symmetry in the first half of the sequence and multiple nonsense codons in all three reading frames. By contrast, the corresponding region in S. aureur consists of 1000 bp capable of forming a similar arrangement of stem-loop structures but with an open reading frame, sited 177 bp downstream of the end of rplL and 217 bp upstream of the beginning of the rpoB gene, with consensus initiation and termination signals, which if translated would generate a 22,665 Da protein with 202 amino acids. In view of the inability to find any significant homology with other proteins in the data bank and because the evidence suggests, as in E. coli, that the rpLrpoB intergenic sequence is involved in regulation it is proposed that the expression product of orf202 may be a further element of control in the S. aureus /I operon.

Research paper thumbnail of What The Papers Say: Conservation of RNA polymerase

Research paper thumbnail of Nucleotide sequence of the Staphylococcus aureus RNA polymerase rpoB gene and comparison of its predicted amino acid sequence with those of other bacteria

Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1995

Tn5405 is a 12 kb staphylococcal composite transposon delimited by two inverted copies of the ins... more Tn5405 is a 12 kb staphylococcal composite transposon delimited by two inverted copies of the insertion sequence IS1 182. This transposon carries two aminoglycosides resistance genes, aphA-3 and aadE, an altered gene similar to sat4 from Campylobacter coli BElG4, and three open reading frames of unknown functions.

Research paper thumbnail of Phenotypic properties of a unique rpoA mutation (phs) of Escherichia coli

Journal of Bacteriology, 1985

The phs mutation of Escherichia coli has been suggested to affect the Na+/H+ antiport (D. Zilbers... more The phs mutation of Escherichia coli has been suggested to affect the Na+/H+ antiport (D. Zilberstein, E. Padan, and S. Schuldiner, FEBS Lett. 168:327-330, 1980). We have recently shown that the mutation affects the rpoA gene and thus affects transcription. The extent of the pleiotropy of the phs mutation was investigated. In addition to the previously reported growth defect on L-glutamate and melibiose, the mutation also affects at least two other metabolic systems. The transport and metabolism of arabinose is impaired and the transport of sulfate is reduced. The extent to which the effects of the phs mutation on metabolism are due to a defect in the Na+/H+ antiport was investigated, and no causal role for this transport system in the metabolic defects was found.

Research paper thumbnail of Genetic studies of the phs locus of Escherichia coli, a mutation causing pleiotropic lesions in metabolism and pH homeostasis

FEBS Letters, 1984

ft is well established that a wide range of bacteria exhibit a remarkable degree of cytoplasmic p... more ft is well established that a wide range of bacteria exhibit a remarkable degree of cytoplasmic pH bomeost~sis despite vexatious in external pH [t,2], However, the molecular m~b~isms which contribute to this ~omeostasis are only vaguely understood [Z]. The development of our understand~ug of many regulatory phenomena in bacteria has been ~o~seq~e~t upon genetic studies [3-s]. Thus, the isoiation of mut~ts defective in pH hom~st~is has been a major aim of workers in the field [6-S]. Of such mutants one of the most im~rtant has been a m~tatjon described as aff~ti~~ the Na'fH" antiport in E~~~~~c~~~ coli f@s) which results in a failure to grow at aika~~ne pH [&-X0], The p&s mutant was isolated after UV rn~~ge~~~s and ~~~~~~~ e~ri~hrn~nf as a stray unable to grow on either meiib~ose or glutamate ]SJ. Both of these carbon sources are believed to be taken up predominantly by Na~~dep~ndent meshes. These systems are dependent upon an Na* gradient ~tabl~shed by the Na*lH" antipo~ (11 3 1 Z]. Loss of the Na*/N" antiport would be ex

Research paper thumbnail of phs Locus of Escherichia coli, a mutation causing pleiotropic lesions in metabolism, is an rpoA allele

Journal of bacteriology, 1985

The phs mutation, which causes a pleiotropic growth defect, has been mapped and shown to be an al... more The phs mutation, which causes a pleiotropic growth defect, has been mapped and shown to be an allele of rpoA, the gene for the alpha subunit of RNA polymerase. The mutation is shown to cause a transcription defect in the arabinose operon, araBAD.

Research paper thumbnail of Refined structure-function analysis of the β-subunit of Escherichia coli RNA polymerase

Biochemical Society Transactions, 1989

Research paper thumbnail of Roles of the trkB and trkC gene products of Escherichia coli in K+ transport

Biochimie, Jan 31, 1985

Summary N Mutations at the trkB andtrkC loci ofEscherichia coli produce an abnormal efflux of K +... more Summary N Mutations at the trkB andtrkC loci ofEscherichia coli produce an abnormal efflux of K +. Tile mutations are partially dominant in diploids and revert frequently by what appears to be intragenic suppression to the null state. Tile mutations can be reverted by insertion of TnlO hlto the mutated gene, and spontaneous revertants are fidly recessive to the mutant allele hi diploids. K* efflux produced by NEM* and by DNP* persists h~ strahls with presumed null mutations, at either locus, hldicathlg neither gene product is the primary target for the effect of these hlhibitors on K* efflux.

Research paper thumbnail of Site-directed mutagenesis of the aromatic amino acid aminotransferase of Escherichia coli

Biochemical Society Transactions, 1989

Research paper thumbnail of d-Alanine transport in Escherichia coli

Biochemical Society Transactions, 1984

Research paper thumbnail of Comparative sequence analysis and predicted phylogeny of the DNA-dependent RNA polymerase beta subunits of Staphylococcus aureus and other eubacteria

Biochemical Society transactions, 1993

Research paper thumbnail of Antigenicity of the Aeromonas salmonicida maltose-inducible outer membrane porin (maltoporin) and a hybrid protein derived from it

Biochemical Society transactions, 1993

Research paper thumbnail of Properties and physiology of energy-coupled mutants of Escherichia coli

Biochemical Society transactions, 1984

Research paper thumbnail of ‘Stop-codon-specific’ restriction endonucleases: their use in mapping and gene manipulation

Gene, 1992

Certain restriction endonucleases recognise target sequences that contain the stop triplet TAG an... more Certain restriction endonucleases recognise target sequences that contain the stop triplet TAG and are commonly either 4 or 6 bp in length. Interestingly, these restriction targets do not occur at the frequency expected on the basis of base composition and size. For example, the tetranucleotide MaeI recognition sequence (CTAG) occurs considerably less commonly (5-8-fold) in the genome of Escherichia coli (and many other eubacteria) than expected from mononucleotide frequencies. This surprising rarity is particularly evident in protein-encoding genes and is largely dictated by codon usage. Thus, amber (TAG) nonsense mutations frequently give rise to novel MaeI (CTAG) sites which are unique within a translated region. Such amber/MaeI sites, whether arising spontaneously or created in vitro by site-directed mutagenesis, act as a useful physical marker for the presence of the nonsense mutation and are a convenient startpoint for a range of diverse procedures. These features provide a useful supplement to protein engineering methods which use nonsense suppression to mediate amino acid replacements.

Research paper thumbnail of Roles of the trkB and trkC gene products of Escherichia coli in K+ transport

Biochimie, 1985

Summary N Mutations at the trkB andtrkC loci ofEscherichia coli produce an abnormal efflux of K +... more Summary N Mutations at the trkB andtrkC loci ofEscherichia coli produce an abnormal efflux of K +. Tile mutations are partially dominant in diploids and revert frequently by what appears to be intragenic suppression to the null state. Tile mutations can be reverted by insertion of TnlO hlto the mutated gene, and spontaneous revertants are fidly recessive to the mutant allele hi diploids. K* efflux produced by NEM* and by DNP* persists h~ strahls with presumed null mutations, at either locus, hldicathlg neither gene product is the primary target for the effect of these hlhibitors on K* efflux.

Research paper thumbnail of What The Papers Say: Conservation of RNA polymerase

Research paper thumbnail of phs Locus of Escherichia coli, a Mutation Causing Pleiotropic Lesions in Metabolism, Is an rpoA Allele

The phs mutation, which causes a pleiotropic growth defect, has been mapped and shown to be an al... more The phs mutation, which causes a pleiotropic growth defect, has been mapped and shown to be an allele of rpoA, the gene for the a subunit of RNA polymerase. The mutation is shown to cause a transcription defect in the arabinose operon, araBAD.