Mutational analysis of Escherichia coli DNA ligase identifies amino acids required for nick-ligation in vitro and for in vivo complementation of the growth of yeast cells deleted for CDC9 and LIG4

RNA (guanine-7-)-methyltransferase is the enzyme responsible for methylating the 5' cap structure of eukaryotic mRNA. The Saccharomyces cerevisiae enzyme is a 436-amino-acid protein encoded by the essential ABD1 gene. In this study, deletion and point mutations in ABD1 were tested for the ability to support growth of an abd1 null strain. Elimination of 109 amino acids from the N terminus had no effect on cell viability, whereas a more extensive N-terminal deletion of 155 residues was lethal, as was a C-terminal deletion of 55 amino acids. Alanine substitution mutations were introduced at eight conserved residues within a 206-amino-acid region of similarity between ABD1 and the methyltransferase domain of the vaccinia virus capping enzyme. ABD1 alleles H253A (encoding a substitution of alanine for histidine at position 253), T282A, E287A, E361A, and Y362A were viable, whereas G174A, D178A, and Y254A were either lethal or severely defective for growth. Alanine-substituted and amino-truncated ABD1 proteins were expressed in bacteria, purified, and tested for cap methyltransferase activity in vitro. Mutations that were viable in yeast cells had either no effect or only a moderate effect on the specific methyltransferase activity of the mutated ABD1 protein, whereas mutations that were deleterious in vivo yielded proteins that were catalytically defective in vitro. These findings substantiate for the first time the long-held presumption that cap methylation is an essential function in eukaryotic cells.

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Important Role for Phylogenetically Invariant PP2Acα Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/156.1.21 ◽

2000 ◽

Vol 156 (1) ◽

pp. 21-29 ◽

Cited By ~ 1

Author(s):

David R H Evans ◽

Brian A Hemmings

Keyword(s):

Protein Interactions ◽

Active Site ◽

Protein Function ◽

Mutational Analysis ◽

Yeast Cells ◽

Temperature Sensitive ◽

Active Site Residues ◽

Catalytic Function

Abstract PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acα functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acα Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acα catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acα C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acα catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

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Mutational Analysis of Residues in PriA and PriC Affecting Their Ability To Interact with SSB in Escherichia coli K-12

Journal of Bacteriology ◽

10.1128/jb.00404-20 ◽

2020 ◽

Vol 202 (23) ◽

Author(s):

Anastasiia N. Klimova ◽

Steven J. Sandler

Keyword(s):

Escherichia Coli ◽

Mutational Analysis ◽

Wild Type ◽

Content Type ◽

Growth Defects ◽

C Terminus ◽

K 12 ◽

And Function

ABSTRACT Escherichia coli PriA and PriC recognize abandoned replication forks and direct reloading of the DnaB replicative helicase onto the lagging-strand template coated with single-stranded DNA-binding protein (SSB). Both PriA and PriC have been shown by biochemical and structural studies to physically interact with the C terminus of SSB. In vitro, these interactions trigger remodeling of the SSB on ssDNA. priA341(R697A) and priC351(R155A) negated the SSB remodeling reaction in vitro. Plasmid-carried priC351(R155A) did not complement priC303::kan, and priA341(R697A) has not yet been tested for complementation. Here, we further studied the SSB-binding pockets of PriA and PriC by placing priA341(R697A), priA344(R697E), priA345(Q701E), and priC351(R155A) on the chromosome and characterizing the mutant strains. All three priA mutants behaved like the wild type. In a ΔpriB strain, the mutations caused modest increases in SOS expression, cell size, and defects in nucleoid partitioning (Par−). Overproduction of SSB partially suppressed these phenotypes for priA341(R697A) and priA344(R697E). The priC351(R155A) mutant behaved as expected: there was no phenotype in a single mutant, and there were severe growth defects when this mutation was combined with ΔpriB. Analysis of the priBC mutant revealed two populations of cells: those with wild-type phenotypes and those that were extremely filamentous and Par− and had high SOS expression. We conclude that in vivo, priC351(R155A) identified an essential residue and function for PriC, that PriA R697 and Q701 are important only in the absence of PriB, and that this region of the protein may have a complicated relationship with SSB. IMPORTANCE Escherichia coli PriA and PriC recruit the replication machinery to a collapsed replication fork after it is repaired and needs to be restarted. In vitro studies suggest that the C terminus of SSB interacts with certain residues in PriA and PriC to recruit those proteins to the repaired fork, where they help remodel it for restart. Here, we placed those mutations on the chromosome and tested the effect of mutating these residues in vivo. The priC mutation completely abolished function. The priA mutations had no effect by themselves. They did, however, display modest phenotypes in a priB-null strain. These phenotypes were partially suppressed by SSB overproduction. These studies give us further insight into the reactions needed for replication restart.

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Mutational analysis of yeast profilin.

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7864 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7864-7873 ◽

Cited By ~ 61

Author(s):

B K Haarer ◽

A S Petzold ◽

S S Brown

Keyword(s):

Amino Acids ◽

Adenylate Cyclase ◽

Mutational Analysis ◽

Actin Binding ◽

In Vitro Assays ◽

C Terminus ◽

Physiological Relevance ◽

In Vivo Effects

We have mutated two regions within the yeast profilin gene in an effort to functionally dissect the roles of actin and phosphatidylinositol 4,5-bisphosphate (PIP2) binding in profilin function. A series of truncations was carried out at the C terminus of profilin, a region that has been implicated in actin binding. Removal of the last three amino acids nearly eliminated the ability of profilin to bind polyproline in vitro but had no dramatic in vivo effects. Thus, the extreme C terminus is implicated in polyproline binding, but the physiological relevance of this interaction is called into question. More extensive truncation, of up to eight amino acids, had in vivo effects of increasing severity and resulted in changes in conformation and expression level of the mutant profilins. However, the ability of these mutants to bind actin in vitro was not eliminated, suggesting that this region cannot be solely responsible for actin binding. We also mutagenized a region of profilin that we hypothesized might be involved in PIP2 binding. Alteration of basic amino acids in this region produced mutant profilins that functioned well in vivo. Many of these mutants, however, were unable to suppress the loss of adenylate cyclase-associated protein (Cap/Srv2p [A. Vojtek, B. Haarer, J. Field, J. Gerst, T. D. Pollard, S. S. Brown, and M. Wigler, Cell 66:497-505, 1991]), indicating that a defect could be demonstrated in vivo. In vitro assays demonstrated that the inability to suppress loss of Cap/Srv2p correlated with a defect in the interaction with actin, independently of whether PIP2 binding was reduced. Since our earlier studies of Acanthamoeba profilins suggested the importance of PIP2 binding for suppression, we conclude that both activities are implicated and that an interplay between PIP2 binding and actin binding may be important for profilin function.

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Acetolactate Synthase from Bacillus subtilis Serves as a 2-Ketoisovalerate Decarboxylase for Isobutanol Biosynthesis in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.01160-09 ◽

2009 ◽

Vol 75 (19) ◽

pp. 6306-6311 ◽

Cited By ~ 65

Author(s):

Shota Atsumi ◽

Zhen Li ◽

James C. Liao

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Bacillus Subtilis ◽

Lactococcus Lactis ◽

Acetolactate Synthase ◽

Side Chains ◽

Decarboxylase Activity ◽

Small Side

ABSTRACTA pathway toward isobutanol production previously constructed inEscherichia coliinvolves 2-ketoacid decarboxylase (Kdc) fromLactococcus lactisthat decarboxylates 2-ketoisovalerate (KIV) to isobutyraldehyde. Here, we showed that a strain lacking Kdc is still capable of producing isobutanol. We found that acetolactate synthase fromBacillus subtilis(AlsS), which originally catalyzes the condensation of two molecules of pyruvate to form 2-acetolactate, is able to catalyze the decarboxylation of KIV like Kdc both in vivo and in vitro. Mutational studies revealed that the replacement of Q487 with amino acids with small side chains (Ala, Ser, and Gly) diminished only the decarboxylase activity but maintained the synthase activity.

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The Escherichia coli Ada Protein Can Interact with Two Distinct Determinants in the ς70 Subunit of RNA Polymerase According to Promoter Architecture: Identification of the Target of Ada Activation at the alkAPromoter

Journal of Bacteriology ◽

10.1128/jb.181.5.1524-1529.1999 ◽

1999 ◽

Vol 181 (5) ◽

pp. 1524-1529 ◽

Cited By ~ 34

Author(s):

Paolo Landini ◽

Stephen J. W. Busby

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Rna Polymerase ◽

Promoter Architecture ◽

Charged Amino Acids ◽

Identify Amino Acid ◽

Ada Protein ◽

Positively Charged

ABSTRACT The methylated form of the Ada protein (meAda) activates transcription from the Escherichia coli ada,aidB, and alkA promoters with different mechanisms. In this study we identify amino acid substitutions in region 4 of the RNA polymerase subunit ς70 that affect Ada-activated transcription at alkA. Substitution to alanine of residues K593, K597, and R603 in ς70 region 4 results in decreased Ada-dependent binding of RNA polymerase to thealkA promoter in vitro and impairs alkAtranscription both in vivo and in vitro, suggesting that these residues define a determinant for meAda-ς70interaction. In a previous study (P. Landini, J. A. Bown, M. R. Volkert, and S. J. W. Busby, J. Biol. Chem. 273:13307–13312, 1998), we showed that a set of negatively charged amino acids in ς70 region 4 is involved inmeAda-ς70 interaction at the adaand aidB promoters. However, the alanine substitutions of positively charged residues K593, K597, and R603 do not affectmeAda-dependent transcription at ada andaidB. Unlike the ς70 amino acids involved in the interaction with meAda at the ada andaidB promoters, K593, K597, and R603 are not conserved in ςS, an alternative ς subunit of RNA polymerase mainly expressed during the stationary phase of growth. WhilemeAda is able to promote transcription by the ςS form of RNA polymerase (EςS) atada and aidB, it fails to do so atalkA. We propose that meAda can activate transcription at different promoters by contacting distinct determinants in ς70 region 4 in a manner dependent on the location of the Ada binding site.

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Mutational Analysis of the MutH Protein fromEscherichia coli

Journal of Biological Chemistry ◽

10.1074/jbc.m007935200 ◽

2000 ◽

Vol 276 (15) ◽

pp. 12113-12119 ◽

Cited By ~ 22

Author(s):

Tamalette Loh ◽

Kenan C. Murphy ◽

Martin G. Marinus

Keyword(s):

Amino Acids ◽

Dna Binding ◽

Mutational Analysis ◽

Site Directed Mutagenesis ◽

Mutant Proteins ◽

Flexible Loop ◽

Loop Regions ◽

Binding Residues

Site-directed mutagenesis was performed on several areas of MutH based on the similarity of MutH andPvuII structural models. The aims were to identify DNA-binding residues; to determine whether MutH has the same mechanism for DNA binding and catalysis asPvuII; and to localize the residues responsible for MutH stimulation by MutL. No DNA-binding residues were identified in the two flexible loop regions of MutH, although similar loops inPvuII are involved in DNA binding. Two histidines in MutH are in a similar position as two histidines (His-84 and His-85) inPvuII that signal for DNA binding and catalysis. These MutH histidines (His-112 and His-115) were changed to alanines, but the mutant proteins had wild-type activity bothin vivoandin vitro. The results indicate that the MutH signal for DNA binding and catalysis remains unknown. Instead, a lysine residue (Lys-48) was found in the first flexible loop that functions in catalysis together with the three presumed catalytic amino acids (Asp-70, Glu-77, and Lys-79). Two deletion mutations (MutHΔ224 and MutHΔ214) in the C-terminal end of the protein, localized the MutL stimulation region to five amino acids (Ala-220, Leu-221, Leu-222, Ala-223, and Arg-224).

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Substrate specificities of Escherichia coli ItaT that acetylates aminoacyl-tRNAs

Nucleic Acids Research ◽

10.1093/nar/gkaa487 ◽

2020 ◽

Cited By ~ 1

Author(s):

Chuqiao Zhang ◽

Yuka Yashiro ◽

Yuriko Sakaguchi ◽

Tsutomu Suzuki ◽

Kozo Tomita

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Amino Group ◽

Hydrophobic Pocket ◽

Hydrophobic Residues ◽

Substrate Specificities ◽

E Coli ◽

Hydrophobic Amino Acids

Abstract Escherichia coli ItaT toxin reportedly acetylates the α-amino group of the aminoacyl-moiety of Ile-tRNAIle specifically, using acetyl-CoA as an acetyl donor, thereby inhibiting protein synthesis. The mechanism of the substrate specificity of ItaT had remained elusive. Here, we present functional and structural analyses of E. coli ItaT, which revealed the mechanism of ItaT recognition of specific aminoacyl-tRNAs for acetylation. In addition to Ile-tRNAIle, aminoacyl-tRNAs charged with hydrophobic residues, such as Val-tRNAVal and Met-tRNAMet, were acetylated by ItaT in vivo. Ile-tRNAIle, Val-tRNAVal and Met-tRNAMet were acetylated by ItaT in vitro, while aminoacyl-tRNAs charged with other hydrophobic residues, such as Ala-tRNAAla, Leu-tRNALeu and Phe-tRNAPhe, were less efficiently acetylated. A comparison of the structures of E. coli ItaT and the protein N-terminal acetyltransferase identified the hydrophobic residues in ItaT that possibly interact with the aminoacyl moiety of aminoacyl-tRNAs. Mutations of the hydrophobic residues of ItaT reduced the acetylation activity of ItaT toward Ile-tRNAIlein vitro, as well as the ItaT toxicity in vivo. Altogether, the size and shape of the hydrophobic pocket of ItaT are suitable for the accommodation of the specific aminoacyl-moieties of aminoacyl-tRNAs, and ItaT has broader specificity toward aminoacyl-tRNAs charged with certain hydrophobic amino acids.

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Structure of Crystallined-Tyr-tRNATyrDeacylase

Journal of Biological Chemistry ◽

10.1074/jbc.m106550200 ◽

2001 ◽

Vol 276 (50) ◽

pp. 47285-47290 ◽

Cited By ~ 42

Author(s):

Maria-Laura Ferri-Fioni ◽

Emmanuelle Schmitt ◽

Julie Soutourina ◽

Pierre Plateau ◽

Yves Mechulam ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Active Sites ◽

Yeast Cells ◽

Crystallographic Structure ◽

Cell Growth Inhibition ◽

E Coli ◽

Sequence Homologies ◽

Β Sheet

Cell growth inhibition by severald-amino acids can be explained by anin vivoproduction ofd-aminoacyl-tRNA molecules.Escherichia coliand yeast cells express an enzyme,d-Tyr-tRNATyrdeacylase, capable of recycling suchd-aminoacyl-tRNA molecules into free tRNA andd-amino acid. Accordingly, upon inactivation of the genes of the above deacylases, the toxicity ofd-amino acids increases. Orthologs of the deacylase are found in many cells. In this study, the crystallographic structure of dimericE. colid-Tyr-tRNATyrdeacylase at 1.55 Å resolution is reported. The structure corresponds to a β-barrel closed on one side by a β-sheet lid. This barrel results from the assembly of the two subunits. Analysis of the structure in relation with sequence homologies in the orthologous family suggests the location of the active sites at the carboxy end of the β-strands. The solved structure markedly differs from those of all other documented tRNA-dependent hydrolases.

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Posttranscriptional Activation of the Transcriptional Activator Rob by Dipyridyl in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.184.5.1407-1416.2002 ◽

2002 ◽

Vol 184 (5) ◽

pp. 1407-1416 ◽

Cited By ~ 57

Author(s):

Judah L. Rosner ◽

Bindi Dangi ◽

Angela M. Gronenborn ◽

Robert G. Martin

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Transcriptional Activator ◽

Terminal Domain ◽

Activation Of Transcription ◽

Transcription In Vitro ◽

Activity State ◽

Low Activity

ABSTRACT The transcriptional activator Rob consists of an N-terminal domain (NTD) of 120 amino acids responsible for DNA binding and promoter activation and a C-terminal domain (CTD) of 169 amino acids of unknown function. Although several thousand molecules of Rob are normally present per Escherichia coli cell, they activate promoters of the rob regulon poorly. We report here that in cells treated with either 2,2"- or 4,4"-dipyridyl (the latter is not a metal chelator), Rob-mediated transcription of various rob regulon promoters was increased substantially. A small, growth-phase-dependent effect of dipyridyl on the rob promoter was observed. However, dipyridyl enhanced Rob's activity even when rob was regulated by a heterologous (lac) promoter showing that the action of dipyridyl is mainly posttranscriptional. Mutants lacking from 30 to 166 of the C-terminal amino acids of Rob had basal levels of activity similar to that of wild-type cells, but dipyridyl treatment did not enhance this activity. Thus, the CTD is not an inhibitor of Rob but is required for activation of Rob by dipyridyl. In contrast to its relatively low activity in vivo, Rob binding to cognate DNA and activation of transcription in vitro is similar to that of MarA, which has a homologous NTD but no CTD. In vitro nuclear magnetic resonance studies demonstrated that 2,2"-dipyridyl binds to Rob but not to the CTD-truncated Rob or to MarA, suggesting that the effect of dipyridyl on Rob is direct. Thus, it appears that Rob can be converted from a low activity state to a high-activity state by a CTD-mediated mechanism in vivo or by purification in vitro.

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