scholarly journals In Vivo Identification of Intermediate Stages of the DNA Inversion Reaction Catalyzed by the Salmonella Hin Recombinase

Genetics ◽  
1998 ◽  
Vol 149 (4) ◽  
pp. 1649-1663
Author(s):  
Oliver Z Nanassy ◽  
Kelly T Hughes
Keyword(s):  
Amino Acids ◽  
Biochemical Analysis ◽  
Mutational Analysis ◽  
Recombination Reaction ◽  
Recombination Site ◽  
Dna Inversion ◽  
Hin Recombinase ◽  
One Step ◽  
A Site

Abstract The Hin recombinase catalyzes a site-specific recombination reaction that results in the reversible inversion of a 1-kbp segment of the Salmonella chromosome. The DNA inversion reaction catalyzed by the Salmonella Hin recombinase is a dynamic process proceeding through many intermediate stages, requiring multiple DNA sites and the Fis accessory protein. Biochemical analysis of this reaction has identified intermediate steps in the inversion reaction but has not yet revealed the process by which transition from one step to another occurs. Because transition from one reaction step to another proceeds through interactions between specific amino acids, and between amino acids and DNA bases, it is possible to study these transitions through mutational analysis of the proteins involved. We isolated a large number of mutants in the Hin recombinase that failed to carry out the DNA exchange reaction. We generated genetic tools that allowed the assignment of these mutants to specific transition steps in the recombination reaction. This genetic analysis, combined with further biochemical analysis, allowed us to define contributions by specific amino acids to individual steps in the DNA inversion reaction. Evidence is also presented in support of a model that Fis protein enhances the binding of Hin to the hixR recombination site. These studies identified regions within the Hin recombinase involved in specific transition steps of the reaction and provided new insights into the molecular details of the reaction mechanism.

scholarly journals Purification and Characterization of the R64 Shufflon-Specific Recombinase

2000 ◽  
Vol 182 (10) ◽  
pp. 2787-2792 ◽  
Author(s):  
Atsuko Gyohda ◽  
Teruya Komano
Keyword(s):  
Electrophoretic Analysis ◽  
Recombination Reaction ◽  
Site Specific ◽  
Dna Inversion ◽  
Repeat Sequences ◽  
In Vitro Recombination ◽  
Recombinase Gene ◽  
A Site

ABSTRACT The shufflon, a multiple DNA inversion system in plasmid R64, consists of four invertible DNA segments which are separated and flanked by seven 19-bp repeat sequences. The product of a site-specific recombinase gene, rci, promotes site-specific recombination between any two of the inverted 19-bp repeat sequences of the shufflon. To analyze the molecular mechanism of this recombination reaction, Rci protein was overproduced and purified. The purified Rci protein promoted the in vitro recombination reaction between the inverted 19-bp repeats of supercoiled DNA of a plasmid carrying segment A of the R64 shufflon. The recombination reaction was enhanced by the bacterial host factor HU. Gel electrophoretic analysis indicated that the Rci protein specifically binds to the DNA segments carrying the 19-bp sequences. The binding affinity of the Rci protein to the four shufflon segments as well as four synthetic 19-bp sequences differed greatly: among the four 19-bp repeat sequences, the repeat-a and -d sequences displayed higher affinity to Rci protein. These results suggest that the differences in the affinity of Rci protein for the 19-bp repeat sequences determine the inversion frequencies of the four segments.

scholarly journals Mutational analysis of yeast profilin.

1993 ◽  
Vol 13 (12) ◽  
pp. 7864-7873 ◽  
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.

scholarly journals Hin Recombinase Mutants Functionally Disrupted in Interactions with Fis

2001 ◽  
Vol 183 (1) ◽  
pp. 28-35 ◽  
Author(s):  
Oliver Z. Nanassy ◽  
Kelly T. Hughes
Keyword(s):  
Dna Binding ◽  
Dna Cleavage ◽  
Genetic Screen ◽  
Recombination Reaction ◽  
Genetic Classification ◽  
Hin Recombinase

ABSTRACT A previous genetic screen was designed to separate Hin recombinase mutants into distinct classes based on the stage in the recombination reaction at which they are blocked (O. Nanassy, Zoltan, and K. T. Hughes, Genetics 149:1649–1663, 1998). One class of DNA binding-proficient, recombination-deficient mutants was predicted by genetic classification to be defective in the step prior to invertasome formation. Based on the genetic criteria, mutants from this class were also inferred to be defective in interactions with Fis. In order to understand how the genetic classification relates to individual biochemical steps in the recombination reaction these mutants, R123Q, T124I, and A126T, were purified and characterized for DNA cleavage and recombination activities. Both the T124I and A126T mutants were partially active, whereas the R123Q mutant was inactive. The A126T mutant was not as defective for recombination as the T124I allele and could be partially rescued for recombination both in vivo and in vitro by increasing the concentration of Fis protein. Rescue of the A126T allele required the Fis protein to be DNA binding proficient. A model for a postsynaptic role for Fis in the inversion reaction is presented.

scholarly journals Mutational Analysis of the Yeast DEAH-Box Splicing Factor Prp16

Genetics ◽  
1998 ◽  
Vol 149 (2) ◽  
pp. 807-815 ◽  
Author(s):  
Hans-Rudolf Hotz ◽  
Beate Schwer
Keyword(s):  
Amino Acids ◽  
Mutational Analysis ◽  
Splicing Factor ◽  
Splicing Factors ◽  
Sequence Motifs ◽  
Wild Type ◽  
Alanine Scanning Mutagenesis ◽  
Alanine Scan ◽  
Hydrolysis Of

Abstract Prp16 is an essential yeast splicing factor that catalyzes RNA-dependent hydrolysis of nucleoside triphosphates. Prp16 is a member of the DEAH-box protein family, which is defined by six collinear sequence motifs. The importance of residues within four of the conserved motifs was assessed by alanine-scanning mutagenesis. Mutant alleles of PRP16 were tested for in vivo function by complementation of a Δprp16 null strain. In motif I (GETGSGKT), alanine substitutions at Gly-378, Lys-379, and Thr-380 were lethal, whereas replacement of the amino acids in positions 373–377 were viable. In the signature DEAH-box (motif II), Asp-473 and Glu-474 were essential, whereas the H476A mutant was viable. The S505A and T507A mutants in motif III (SAT) were viable. In motif VI (QRSGRAGRTAPG), mutants Q685A, R686A, G688A, R689A, and R692A were lethal, whereas G691A, P695A, and G696A supported growth. Instructive structure-function relationships were established by conservative substitutions at essential residues identified by alanine scan. Overexpression of nonviable alleles impaired the growth of wild-type PRP16 cells. Deletion analysis of the 1071-amino-acid Prp16 protein revealed that the N-terminal 204 amino acids and the C-terminal 100 residues were dispensable for PRP16 function in vivo. These studies provide an instructive framework for functional analysis of other DEAH-box splicing factors.

scholarly journals Mutational Analysis of the MutH Protein fromEscherichia coli

2000 ◽  
Vol 276 (15) ◽  
pp. 12113-12119 ◽  
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).

scholarly journals Measurement of average decoding rates of the 61 sense codons in vivo

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Justin Gardin ◽  
Rukhsana Yeasmin ◽  
Alisa Yurovsky ◽  
Ying Cai ◽  
Steve Skiena ◽  
...  
Keyword(s):  
Amino Acids ◽  
Ribosome Profiling ◽  
High Coverage ◽  
Peptide Bonds ◽  
Synonymous Codons ◽  
Specific Effects ◽  
Exit Tunnel ◽  
A Site ◽  
Novel Algorithm

Most amino acids can be encoded by several synonymous codons, which are used at unequal frequencies. The significance of unequal codon usage remains unclear. One hypothesis is that frequent codons are translated relatively rapidly. However, there is little direct, in vivo, evidence regarding codon-specific translation rates. In this study, we generate high-coverage data using ribosome profiling in yeast, analyze using a novel algorithm, and deduce events at the A- and P-sites of the ribosome. Different codons are decoded at different rates in the A-site. In general, frequent codons are decoded more quickly than rare codons, and AT-rich codons are decoded more quickly than GC-rich codons. At the P-site, proline is slow in forming peptide bonds. We also apply our algorithm to short footprints from a different conformation of the ribosome and find strong amino acid-specific (not codon-specific) effects that may reflect interactions with the exit tunnel of the ribosome.

scholarly journals Mutational analysis of the Saccharomyces cerevisiae ABD1 gene: cap methyltransferase activity is essential for cell growth.

1996 ◽  
Vol 16 (2) ◽  
pp. 475-480 ◽  
Author(s):  
X Mao ◽  
B Schwer ◽  
S Shuman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Mutational Analysis ◽  
Point Mutations ◽  
Yeast Cells ◽  
Terminal Deletion ◽  
Methyltransferase Activity

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.

scholarly journals Fat body: a site of hemoglobin synthesis in Chironomus thummi (diptera).

1976 ◽  
Vol 69 (2) ◽  
pp. 264-274 ◽  
Author(s):  
G Bergtrom ◽  
H Laufer ◽  
R Rogers
Keyword(s):  
Amino Acids ◽  
Salivary Glands ◽  
Culture Medium ◽  
Aminolevulinic Acid ◽  
Fat Body ◽  
Hemoglobin Synthesis ◽  
A Site ◽  
Coomassie Brilliant ◽  
Chironomus Thummi

Fourth instar larvae of Chironomus thummi were permitted to incorporate labeled amino acids and/or sigma-aminolevulinic acid (sigma-ALA) in vivo and in organ culture. The products secreted into the hemolymph or into the culture medium were examined by acrylamide gel electrophoresis. Nine electrophoretic bands can be resolved as hemoglobins without staining. When gels are sliced for scintillation counting, incorporated amino acids and sigma-ALA are shown to be associated primarily with the same nine hemoglobin bands, suggesting that hemoglobins are assembled and secreted. Staining of gels with Coomassie brilliant blue reveals that there are several bands in addition to the visible hemoglobins. These bands incorporate amino acids, but not sigma-ALA, suggesting that they are non-heme proteins. The results of culturing isolated salivary glands, gut, and fat body demonstrate that the fat body is the major site of hemoglobin synthesis and secretion. Labeled products of the gut represent about 5% of the total hemoglobins produced by the tissues, while no hemoglobins are produced by the salivary glands. Although nine hemoglobins are visibly resolved on gels, labeling techniques reveal as many as 14 hemoglobins. This is the first demonstration of hemoglobin synthesis by specific tissues in culture in an invertebrate.

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