scholarly journals Different Rifampin Sensitivities of Escherichia coli and Mycobacterium tuberculosis RNA Polymerases Are Not Explained by the Difference in the β-Subunit Rifampin Regions I and II

2005 ◽  
Vol 49 (4) ◽  
pp. 1587-1590 ◽  
Author(s):  
N. Zenkin ◽  
A. Kulbachinskiy ◽  
I. Bass ◽  
V. Nikiforov
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Mycobacterium Tuberculosis ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Rna Polymerase ◽  
Β Subunit ◽  
E Coli ◽  
Increased Sensitivity ◽  
The Difference

ABSTRACT Mycobacterium tuberculosis RNA polymerase is 1,000-fold more sensitive to rifampin than Escherichia coli RNA polymerase. Chimeric E. coli RNA polymerase in which the β-subunit segment encompassing rifampin regions I and II (amino acids [aa] 463 through 590) was replaced with the corresponding region from M. tuberculosis (aa 382 through 509) did not show an increased sensitivity to the antibiotic. Thus, the difference in amino acid sequence between the rifampin regions I and II of the two species does not account for the difference in rifampin sensitivity of the two polymerases.

scholarly journals Association of ω with the C-Terminal Region of the β′ Subunit Is Essential for Assembly of RNA Polymerase in Mycobacterium tuberculosis

2018 ◽  
Vol 200 (12) ◽  
Author(s):  
Chunyou Mao ◽  
Yan Zhu ◽  
Pei Lu ◽  
Lipeng Feng ◽  
Shiyun Chen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mycobacterium Tuberculosis ◽  
Rna Polymerase ◽  
Essential Role ◽  
Β Subunit ◽  
Content Type ◽  
E Coli ◽  
Core Enzyme ◽  
Loop Region

ABSTRACT The ω subunit is the smallest subunit of bacterial RNA polymerase (RNAP). Although homologs of ω are essential in both eukaryotes and archaea, this subunit has been known to be dispensable for RNAP in Escherichia coli and in other bacteria. In this study, we characterized an indispensable role of the ω subunit in Mycobacterium tuberculosis . Unlike the well-studied E. coli RNAP, the M. tuberculosis RNAP core enzyme cannot be functionally assembled in the absence of the ω subunit. Importantly, substitution of M. tuberculosis ω with ω subunits from E. coli or Thermus thermophilus cannot restore the assembly of M. tuberculosis RNAP. Furthermore, by replacing different regions in M. tuberculosis ω with the corresponding regions from E. coli ω, we found a nonconserved loop region in M. tuberculosis ω essential for its function in RNAP assembly. From RNAP structures, we noticed that the location of the C-terminal region of the β′ subunit (β′CTD) in M. tuberculosis RNAP but not in E. coli or T. thermophilus RNAP is close to the ω loop region. Deletion of this β′CTD in M. tuberculosis RNAP destabilized the binding of M. tuberculosis ω on RNAP and compromised M. tuberculosis core assembly, suggesting that these two regions may function together to play a role in ω-dependent RNAP assembly in M. tuberculosis . Sequence alignment of the ω loop and the β′CTD regions suggests that the essential role of ω is probably restricted to mycobacteria. Together, our study characterized an essential role of M. tuberculosis ω and highlighted the importance of the ω loop region in M. tuberculosis RNAP assembly. IMPORTANCE DNA-dependent RNA polymerase (RNAP), which consists of a multisubunit core enzyme (α 2 ββ′ω) and a dissociable σ subunit, is the only enzyme in charge of transcription in bacteria. As the smallest subunit, the roles of ω remain the least well studied. In Escherichia coli and some other bacteria, the ω subunit is known to be nonessential for RNAP. In this study, we revealed an essential role of the ω subunit for RNAP assembly in the human pathogen Mycobacterium tuberculosis , and a mycobacterium-specific ω loop that plays a role in this function was also characterized. Our study provides fresh insights for further characterizing the roles of bacterial ω subunit.

scholarly journals Roles of the C-Terminal Amino Acids of Non-Hexameric Helicases: Insights from Escherichia coli UvrD

2021 ◽  
Vol 22 (3) ◽  
pp. 1018
Author(s):  
Hiroaki Yokota
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Single Molecule ◽  
Underlying Mechanism ◽  
Amino Acid Sequences ◽  
E Coli ◽  
X Ray Crystallography ◽  
Terminal Amino

Helicases are nucleic acid-unwinding enzymes that are involved in the maintenance of genome integrity. Several parts of the amino acid sequences of helicases are very similar, and these quite well-conserved amino acid sequences are termed “helicase motifs”. Previous studies by X-ray crystallography and single-molecule measurements have suggested a common underlying mechanism for their function. These studies indicate the role of the helicase motifs in unwinding nucleic acids. In contrast, the sequence and length of the C-terminal amino acids of helicases are highly variable. In this paper, I review past and recent studies that proposed helicase mechanisms and studies that investigated the roles of the C-terminal amino acids on helicase and dimerization activities, primarily on the non-hexermeric Escherichia coli (E. coli) UvrD helicase. Then, I center on my recent study of single-molecule direct visualization of a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C) used in studies proposing the monomer helicase model. The study demonstrated that multiple UvrDΔ40C molecules jointly participated in DNA unwinding, presumably by forming an oligomer. Thus, the single-molecule observation addressed how the C-terminal amino acids affect the number of helicases bound to DNA, oligomerization, and unwinding activity, which can be applied to other helicases.

scholarly journals d-alanine: d-alanine ligase of Escherichia coli. Expression, purification and inhibitory studies on the cloned enzyme

1992 ◽  
Vol 282 (3) ◽  
pp. 747-752 ◽  
Author(s):  
O A M al-Bar ◽  
C D O'Connor ◽  
I G Giles ◽  
M Akhtar
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Gene Sequence ◽  
Phosphinic Acid ◽  
Mature Protein ◽  
Bamhi Fragment ◽  
E Coli ◽  
Escherichia Coli Expression ◽  
Slow Binding Inhibitor

A 1.2 kb BamHI fragment from pDK30 [Robinson, Kenan, Sweeney & Donachie (1986) J. Bacteriol. 167, 809-817] was cloned in pDOC55 [O'Connor & Timmis (1987) J. Bacteriol. 169, 4457-4482] to give two constructs, pDOC89 and pDOC87, in which the Escherichia coli D-alanine:D-alanine ligase (EC 6.3.2.4) gene (ddl) was placed under the control of the lac and lambda PL promoters respectively. Both constructs, when used to transform E. coli M72, gave similar levels of expression of the ddl gene. The expressed enzyme was purified to homogeneity and the amino acid sequence of its N-terminal region was found to be consistent with that predicted from the gene sequence, except that the N-terminal methionine was not present in the mature protein. [1(S)-Aminoethyl][(2RS)2-carboxy-1-octyl]phosphinic acid (I), previously shown to bind tightly to Enterococcus faecalis and Salmonella typhimurium D-alanine:D-alanine ligases following phosphorylation Parsons, Patchett, Bull, Schoen, Taub, Davidson, Combs, Springer, Gadebusch, Weissberger, Valiant, Mellin & Busch (1988) J. Med. Chem. 31, 1772-1778; Duncan & Walsh (1988) Biochemistry 27, 3709-3714], was found to be a classical slow-binding inhibitor of the E. coli ligase.

Sequence mutations in teleost cardiac troponin C that are permissive of high Ca2+ affinity of site II

2003 ◽  
Vol 284 (5) ◽  
pp. C1176-C1184 ◽  
Author(s):  
Todd E. Gillis ◽  
Chris D. Moyes ◽  
Glen F. Tibbits
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Cardiac Troponin ◽  
Troponin C ◽  
Fluorescent Reporter ◽  
Cardiac Troponin C ◽  
The Difference ◽  
Higher Sensitivity

Cardiac myofibrils isolated from trout heart have been demonstrated to have a higher sensitivity for Ca2+ than mammalian cardiac myofibrils. Using cardiac troponin C (cTnC) cloned from trout and mammalian hearts, we have previously demonstrated that this comparatively high Ca2+ sensitivity is due, in part, to trout cTnC (ScTnC) having twice the Ca2+ affinity of mammalian cTnC (McTnC) over a broad range of temperatures. The amino acid sequence of ScTnC is 92% identical to McTnC. To determine the residues responsible for the high Ca2+ affinity, the function of a number of ScTnC and McTnC mutants was characterized by monitoring an intrinsic fluorescent reporter that monitors Ca2+ binding to site II (F27W). The removal of the COOH terminus (amino acids 90–161) from ScTnC and McTnC maintained the difference in Ca2+ affinity between the truncated cTnC isoforms (ScNTnC and McNTnC). The replacement of Gln29 and Asp30 in ScNTnC with the corresponding residues from McNTnC, Leu and Gly, respectively, reduced Ca2+ affinity to that of McNTnC. These results demonstrate that Gln29 and Asp30 in ScTnC are required for the high Ca2+ affinity of site II.

Molecular Characterization of Genes Coding for Wild-Type and Sulfonylurea-Resistant Acetolactate Synthase in the Cyanobacterium Synechococcus PC C7942

1990 ◽  
Vol 45 (5) ◽  
pp. 538-543 ◽  
Author(s):  
D. Friedberg ◽  
J. Seijffers
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Characterization ◽  
Amino Acid Level ◽  
Acetolactate Synthase ◽  
Mutant Gene ◽  
Wild Type ◽  
E Coli

We present here the isolation and molecular characterization of acetolactate synthase (ALS) genes from the cyanobacterium Synechococcus PCC7942 which specify a sulfonylurea-sensitive enzyme and from the sulfonylurea-resistant mutant SM3/20, which specify resistance to sulfonylurea herbicides. The ALS gene was cloned and mapped by complementation of an Escherichia coli ilv auxotroph that requires branched-chain amino acids for growth and lacks ALS activity. The cyanobacterial gene is efficiently expressed in this heterologous host. The ALS gene codes for 612 amino acids and shows high sequence homology (46%) at the amino acid level with ALS III of E. coli and with the tobacco ALS. The resistant phenotype is a consequence of proline to serine substitution in residue 115 of the deduced amino acid sequence. Functional expression of the mutant gene in wild-type Synechococcus and in E. coli confirmed that this amino-acid substitution is responsible for the resistance. Yet the deduced amino-acid sequence as compared with othjer ALS proteins supports the notion that the amino-acid context of the substitution is important for the resistance.

scholarly journals Cloning, Nucleotide Sequence, and Overexpression of the l-Rhamnose Isomerase Gene from Pseudomonas stutzeri in Escherichia coli

2004 ◽  
Vol 70 (6) ◽  
pp. 3298-3304 ◽  
Author(s):  
Khim Leang ◽  
Goro Takada ◽  
Akihiro Ishimura ◽  
Masashi Okita ◽  
Ken Izumori
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
Pseudomonas Stutzeri ◽  
Fold Increase ◽  
E Coli ◽  
Apparent Homogeneity ◽  
Volumetric Yield

ABSTRACT The gene encoding l-rhamnose isomerase (l-RhI) from Pseudomonas stutzeri was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA responsible for the l-RhI gene revealed an open reading frame of 1,290 bp coding for a protein of 430 amino acid residues with a predicted molecular mass of 46,946 Da. A comparison of the deduced amino acid sequence with sequences in relevant databases indicated that no significant homology has previously been identified. An amino acid sequence alignment, however, suggested that the residues involved in the active site of l-RhI from E. coli are conserved in that from P. stutzeri. The l-RhI gene was then overexpressed in E. coli cells under the control of the T5 promoter. The recombinant clone, E. coli JM109, produced significant levels of l-RhI activity, with a specific activity of 140 U/mg and a volumetric yield of 20,000 U of soluble enzyme per liter of medium. This reflected a 20-fold increase in the volumetric yield compared to the value for the intrinsic yield. The recombinant l-RhI protein was purified to apparent homogeneity on the basis of three-step chromatography. The purified recombinant enzyme showed a single band with an estimated molecular weight of 42,000 in a sodium dodecyl sulfate-polyacrylamide gel. The overall enzymatic properties of the purified recombinant l-RhI protein were the same as those of the authentic one, as the optimal activity was measured at 60�C within a broad pH range from 5.0 to 11.0, with an optimum at pH 9.0.

scholarly journals Bacterial Two-Hybrid Analysis of Interactions between Region 4 of the ς70 Subunit of RNA Polymerase and the Transcriptional Regulators Rsd from Escherichia coli and AlgQ from Pseudomonas aeruginosa

2001 ◽  
Vol 183 (21) ◽  
pp. 6413-6421 ◽  
Author(s):  
Simon L. Dove ◽  
Ann Hochschild
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Rna Polymerase ◽  
Amino Acid Substitution ◽  
Transcriptional Regulators ◽  
Binding Motif ◽  
E Coli ◽  
Two Hybrid

ABSTRACT A number of transcriptional regulators mediate their effects through direct contact with the ς70 subunit ofEscherichia coli RNA polymerase (RNAP). In particular, several regulators have been shown to contact a C-terminal portion of ς70 that harbors conserved region 4. This region of ς contains a putative helix-turn-helix DNA-binding motif that contacts the −35 element of ς70-dependent promoters directly. Here we report the use of a recently developed bacterial two-hybrid system to study the interaction between the putative anti-ς factor Rsd and the ς70 subunit of E. coli RNAP. Using this system, we found that Rsd can interact with an 86-amino-acid C-terminal fragment of ς70 and also that amino acid substitution R596H, within region 4 of ς70, weakens this interaction. We demonstrated the specificity of this effect by showing that substitution R596H does not weaken the interaction between ς and two other regulators shown previously to contact region 4 of ς70. We also demonstrated that AlgQ, a homolog of Rsd that positively regulates virulence gene expression inPseudomonas aeruginosa, can contact the C-terminal region of the ς70 subunit of RNAP from this organism. We found that amino acid substitution R600H in ς70 fromP. aeruginosa, corresponding to the R596H substitution in E. coli ς70, specifically weakens the interaction between AlgQ and ς70. Taken together, our findings suggest that Rsd and AlgQ contact similar surfaces of RNAP present in region 4 of ς70 and probably regulate gene expression through this contact.

scholarly journals Expression in Escherichia coli and characterization of a reconstituted recombinant 7Fe ferredoxin from Desulfovibrio africanus

1996 ◽  
Vol 314 (1) ◽  
pp. 63-71 ◽  
Author(s):  
Johanneke L. H. BUSCH ◽  
Jacques L. J. BRETON ◽  
Barry M. BARTLETT ◽  
Richard JAMES ◽  
E. Claude HATCHIKIAN ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Magnetic Circular Dichroism ◽  
Wild Type ◽  
E Coli ◽  
Excess Iron ◽  
Expression In Escherichia Coli ◽  
Type D

Desulfovibrio africanus ferredoxin III is a monomeric protein (molecular mass of 6585 Da) that contains one [3Fe-4S]1+/0 and one [4Fe-4S]2+/1+ cluster when isolated aerobically. The amino acid sequence consists of 61 amino acids, including seven cysteine residues that are all involved in co-ordination to the clusters. In order to isolate larger quantities of D. africanus ferredoxin III, we have overexpressed it in Escherichia coli by constructing a synthetic gene based on the amino acid sequence of the native protein. The recombinant ferredoxin was expressed in E. coli as an apoprotein. We have reconstituted the holoprotein by incubating the apoprotein with excess iron and sulphide in the presence of a reducing agent. The reconstituted recombinant ferredoxin appeared to have a lower stability than that of wild-type D. africanus ferredoxin III. We have shown by low-temperature magnetic circular dichroism and EPR spectroscopy that the recombinant ferredoxin contains a [3Fe-4S]1+/0 and a [4Fe-4S]2+/1+ cluster similar to those found in native D. africanus ferredoxin III. These results indicate that the two clusters have been correctly inserted into the recombinant ferredoxin.

scholarly journals Plasmid-Encoded asp Operon Confers a Proton Motive Metabolic Cycle Catalyzed by an Aspartate-Alanine Exchange Reaction

2002 ◽  
Vol 184 (11) ◽  
pp. 2906-2913 ◽  
Author(s):  
Keietsu Abe ◽  
Fumito Ohnishi ◽  
Kyoko Yagi ◽  
Tasuku Nakajima ◽  
Takeshi Higuchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Sequence Analysis ◽  
Amino Acid Sequence ◽  
Expression Vector ◽  
Alcaligenes Faecalis ◽  
E Coli ◽  
Cell Extracts ◽  
Membrane Spanning ◽  
Metabolic Cycle

ABSTRACT Tetragenococcus halophila D10 catalyzes the decarboxylation of l-aspartate with nearly stoichiometric release of l-alanine and CO2. This trait is encoded on a 25-kb plasmid, pD1. We found in this plasmid a putative asp operon consisting of two genes, which we designated aspD and aspT, encoding an l-aspartate-β-decarboxylase (AspD) and an aspartate-alanine antiporter (AspT), respectively, and determined the nucleotide sequences. The sequence analysis revealed that the genes of the asp operon in pD1 were in the following order: promoter → aspD → aspT. The deduced amino acid sequence of AspD showed similarity to the sequences of two known l-aspartate-β-decarboxylases from Pseudomonas dacunhae and Alcaligenes faecalis. Hydropathy analyses suggested that the aspT gene product encodes a hydrophobic protein with multiple membrane-spanning regions. The operon was subcloned into the Escherichia coli expression vector pTrc99A, and the two genes were cotranscribed in the resulting plasmid, pTrcAsp. Expression of the asp operon in E. coli coincided with appearance of the capacity to catalyze the decarboxylation of aspartate to alanine. Histidine-tagged AspD (AspDHis) was also expressed in E. coli and purified from cell extracts. The purified AspDHis clearly exhibited activity of l-aspartate-β-decarboxylase. Recombinant AspT was solubilized from E. coli membranes and reconstituted in proteoliposomes. The reconstituted AspT catalyzed self-exchange of aspartate and electrogenic heterologous exchange of aspartate with alanine. Thus, the asp operon confers a proton motive metabolic cycle consisting of the electrogenic aspartate-alanine antiporter and the aspartate decarboxylase, which keeps intracellular levels of alanine, the countersubstrate for aspartate, high.

scholarly journals Characteristics of a New Enantioselective Thermostable Dipeptidase from Brevibacillus borstelensis BCS-1 and Its Application to Synthesis of a d-Amino-Acid-Containing Dipeptide

2004 ◽  
Vol 70 (3) ◽  
pp. 1570-1575 ◽  
Author(s):  
Dae Heoun Baek ◽  
Jae Jun Song ◽  
Seok-Joon Kwon ◽  
Chung Park ◽  
Chang-Min Jung ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Nucleotide Sequence ◽  
Amino Acid Sequence ◽  
Open Reading Frame ◽  
Nucleotide Sequence Analysis ◽  
Reading Frame ◽  
E Coli ◽  
Specificity Constant ◽  
Optimal Ph

ABSTRACT A new thermostable dipeptidase gene was cloned from the thermophile Brevibacillus borstelensis BCS-1 by genetic complementation of the d-Glu auxotroph Escherichia coli WM335 on a plate containing d-Ala-d-Glu. Nucleotide sequence analysis revealed that the gene included an open reading frame coding for a 307-amino-acid sequence with an M r of 35,000. The deduced amino acid sequence of the dipeptidase exhibited 52% similarity with the dipeptidase from Listeria monocytogenes. The enzyme was purified to homogeneity from recombinant E. coli WM335 harboring the dipeptidase gene from B. borstelensis BCS-1. Investigation of the enantioselectivity (E) to the P1 and P1′ site of Ala-Ala revealed that the ratio of the specificity constant (k cat /Km ) for l-enantioselectivity to the P1 site of Ala-Ala was 23.4 � 2.2 [E = (k cat /Km ) l,d /(k cat /Km ) d,d ], while the d-enantioselectivity to the P1′ site of Ala-Ala was 16.4 � 0.5 [E = (k cat /Km ) l,d /(k cat /Km ) l,l ] at 55�C. The enzyme was stable up to 55�C, and the optimal pH and temperature were 8.5 and 65�C, respectively. The enzyme was able to hydrolyze l-Asp-d-Ala, l-Asp-d-AlaOMe, Z-d-Ala-d-AlaOBzl, and Z-l-Asp-d-AlaOBzl, yet it could not hydrolyze d-Ala-l-Asp, d-Ala-l-Ala, d-AlaNH2, and l-AlaNH2. The enzyme also exhibited β-lactamase activity similar to that of a human renal dipeptidase. The dipeptidase successfully synthesized the precursor of the dipeptide sweetener Z-l-Asp-d-AlaOBzl.

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