scholarly journals Lambda repressor recognizes the approximately 2-fold symmetric half-operator sequences asymmetrically

1989 ◽  
Vol 86 (17) ◽  
pp. 6513-6517 ◽  
Author(s):  
A Sarai ◽  
Y Takeda
Keyword(s):  
Amino Acids ◽  
Symmetric Operator ◽  
Dna Bases ◽  
Base Substitution ◽  
Base Pairs ◽  
Lambda Repressor ◽  
Base Substitutions ◽  
Protein Dna Interactions ◽  
Operator Sequence ◽  
Recognition Code

Results of systematic base-substitution experiments suggest that the lambda repressor dimer, made of identical subunits, recognizes the "pseudo(2-fold)symmetric" operator sequence asymmetrically. Base substitutions within the consensus half of the operator affect binding more than base substitutions within the nonconsensus half of the operator. Furthermore, changing the nonconsensus base pairs to the consensus base pairs does not increase, but decreases, binding. Evidently, the two subunits of the lambda repressor dimer bind to the two halves of the operator differently. This is consistent with the recently determined crystal structure of the complex, which shows that the relative positioning of the amino acids to the DNA bases are slightly different in the two halves of the operator. The sequence-specific interactions indicated by the systematic base-substitution experiments correlate well with the locations of the specific contacts found in the complex. Thus, the amino acids of lambda repressor, mainly of alpha 3-helix and the N-terminus arm, seem to directly read-out the DNA sequence by forming specific hydrogen bonds and hydrophobic contacts to the DNA bases. The observed asymmetric recognition suggests that no recognition code governs amino acids and DNA bases in protein-DNA interactions.

scholarly journals Phage lambda Cro protein and cI repressor use two different patterns of specific protein-DNA interactions to achieve sequence specificity in vivo.

Genetics ◽  
1989 ◽  
Vol 121 (1) ◽  
pp. 5-12
Author(s):  
N Benson ◽  
P Youderian
Keyword(s):  
Specific Protein ◽  
Specific Interactions ◽  
Sequence Specificity ◽  
Base Pairs ◽  
Lambda Repressor ◽  
Protein Dna Interactions ◽  
Cro Protein ◽  
Data Call ◽  
Half Site

Abstract By assaying the binding of wild-type Cro to a set of 40 mutant lambda operators in vivo, we have determined that the 14 outermost base pairs of the 17 base pair, consensus lambda operator are critical for Cro binding. Cro protein recognizes 4 base pairs in a lambda operator half-site in different ways than cI repressor. The sequence determinants of Cro binding at these critical positions in vivo are nearly perfectly consistent with the model proposed by W. F. ANDERSON, D. H. OHLENDORF, Y. TAKEDA and B. W. MATTHEWS and modified by Y. TAKEDA, A. SARAI and V. M. RIVERA for the specific interactions between Cro and its operator, and explain the relative order of affinities of the six natural lambda operators for Cro. Our data call into question the idea that lambda repressor and Cro protein recognize the consensus lambda operator by nearly identical patterns of specific interactions.

scholarly journals Evolution of the genetic code; Evidence from serine codon use disparity inEscherichia coli

2020 ◽  
Vol 117 (46) ◽  
pp. 28572-28575
Author(s):  
Masayori Inouye ◽  
Risa Takino ◽  
Yojiro Ishida ◽  
Keiko Inouye
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Glutamic Acid ◽  
The Other ◽  
Base Substitution ◽  
Inescherichia Coli ◽  
Single Base ◽  
Base Substitutions ◽  
The Origin Of Life ◽  
Codon Use

Among the 20 amino acids, three of them—leucine (Leu), arginine (Arg), and serine (Ser)—are encoded by six different codons. In comparison, all of the other 17 amino acids are encoded by either 4, 3, 2, or 1 codon. Peculiarly, Ser is separated into two disparate Ser codon boxes, differing by at least two-base substitutions, in contrast to Leu and Arg, of which codons are mutually exchangeable by a single-base substitution. We propose that these two different Ser codons independently emerged during evolution. In this hypothesis, at the time of the origin of life there were only seven primordial amino acids: Valine (coded by GUX [X = U, C, A or G]), alanine (coded by GCX), aspartic acid (coded by GAY [Y = U or C]), glutamic acid (coded by GAZ [Z = A or G]), glycine (coded by GGX), Ser (coded by AGY), and Arg (coded by CGX and AGZ). All of these were derived from GGX for glycine by single-base substitutions. Later in evolution, another class of Ser codons, UCX, were derived from alanine codons, GCX, distinctly different from the other primordial Ser codon, AGY. From the analysis of theEscherichia coligenome, we find extensive disparities in the usage of these two Ser codons, as some genes use only AGY for Ser in their genes. In contrast, others use only UCX, pointing to distinct differences in their origins, consistent with our hypothesis.

scholarly journals PCR-Oligonucleotide Ligation Assay for Detection of Point Mutations Associated with Quinolone Resistance in Streptococcus pneumoniae

2003 ◽  
Vol 47 (4) ◽  
pp. 1456-1459 ◽  
Author(s):  
Mai-Ha Bui ◽  
Gregory G. Stone ◽  
Angela M. Nilius ◽  
Laurel Almer ◽  
Robert K. Flamm
Keyword(s):  
Amino Acids ◽  
Streptococcus Pneumoniae ◽  
Sequence Data ◽  
Point Mutations ◽  
Quinolone Resistance ◽  
Clinical Isolates ◽  
Dna Bases ◽  
Single Base ◽  
Base Substitutions ◽  
Oligonucleotide Ligation Assay

ABSTRACT We have developed a PCR-oligonucleotide ligation assay to rapidly identify base substitutions in topoisomerase genes that are associated with quinolone resistance in clinical isolates of Streptococcus pneumoniae. Thirty-seven strains for which the ciprofloxacin MICs were ≥4 μg/ml and 16 strains for which the MICs were ≤2 μg/ml were assayed. Compared with sequence data, the assay correctly identified the DNA bases that encoded amino acids at the four positions most commonly associated with quinolone resistance (Ser79 and Asp83 of ParC and Ser81 and Glu85 of GyrA). Therefore, this procedure can rapidly distinguish single base substitutions associated with quinolone-resistant topoisomerases in S. pneumoniae.

scholarly journals Formation of the Codon Degeneracy during Interdependent Development between Metabolism and Replication

Genes ◽  
2021 ◽  
Vol 12 (12) ◽  
pp. 2023
Author(s):  
Dirson Jian Li
Keyword(s):  
Amino Acids ◽  
Genetic Code ◽  
Driving Force ◽  
Sequence Evolution ◽  
Base Pairs ◽  
Random Sequences ◽  
Relative Stabilities ◽  
Base Substitutions ◽  
Codon Degeneracy

Nirenberg’s genetic code chart shows a profound correspondence between codons and amino acids. The aim of this article is to try to explain the primordial formation of the codon degeneracy. It remains a puzzle how informative molecules arose from the supposed prebiotic random sequences. If introducing an initial driving force based on the relative stabilities of triplex base pairs, the prebiotic sequence evolution became innately nonrandom. Thus, the primordial assignment of the 64 codons to the 20 amino acids has been explained in detail according to base substitutions during the coevolution of tRNAs with aaRSs; meanwhile, the classification of aaRSs has also been explained.

Developing a Genetic System in Deinococcus radiodurans for Analyzing Mutations

Genetics ◽  
2004 ◽  
Vol 166 (2) ◽  
pp. 661-668
Author(s):  
Mandy Kim ◽  
Erika Wolff ◽  
Tiffany Huang ◽  
Lilit Garibyan ◽  
Ashlee M Earl ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Deinococcus Radiodurans ◽  
Genetic System ◽  
Base Change ◽  
Base Substitution ◽  
Wild Type ◽  
Base Pairs ◽  
E Coli ◽  
Radiation Induced ◽  
Induced Mutagenesis

Abstract We have applied a genetic system for analyzing mutations in Escherichia coli to Deinococcus radiodurans, an extremeophile with an astonishingly high resistance to UV- and ionizing-radiation-induced mutagenesis. Taking advantage of the conservation of the β-subunit of RNA polymerase among most prokaryotes, we derived again in D. radiodurans the rpoB/Rif r system that we developed in E. coli to monitor base substitutions, defining 33 base change substitutions at 22 different base pairs. We sequenced >250 mutations leading to Rif r in D. radiodurans derived spontaneously in wild-type and uvrD (mismatch-repair-deficient) backgrounds and after treatment with N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and 5-azacytidine (5AZ). The specificities of NTG and 5AZ in D. radiodurans are the same as those found for E. coli and other organisms. There are prominent base substitution hotspots in rpoB in both D. radiodurans and E. coli. In several cases these are at different points in each organism, even though the DNA sequences surrounding the hotspots and their corresponding sites are very similar in both D. radiodurans and E. coli. In one case the hotspots occur at the same site in both organisms.

In Vivo Consequences of Putative Active Site Mutations in Yeast DNA Polymerases α, ε, δ, and ζ

Genetics ◽  
2001 ◽  
Vol 159 (1) ◽  
pp. 47-64 ◽  
Author(s):  
Youri I Pavlov ◽  
Polina V Shcherbakova ◽  
Thomas A Kunkel
Keyword(s):  
Active Site ◽  
Dna Polymerases ◽  
Base Substitution ◽  
Loss Of Function ◽  
Chromosomal Dna ◽  
Strong Base ◽  
Dna Mismatch ◽  
Catalytic Function ◽  
Base Substitutions

Abstract Several amino acids in the active site of family A DNA polymerases contribute to accurate DNA synthesis. For two of these residues, family B DNA polymerases have conserved tyrosine residues in regions II and III that are suggested to have similar functions. Here we replaced each tyrosine with alanine in the catalytic subunits of yeast DNA polymerases α, δ, ε, and ζ and examined the consequences in vivo. Strains with the tyrosine substitution in the conserved SL/MYPS/N motif in region II in Polδ or Polε are inviable. Strains with same substitution in Rev3, the catalytic subunit of Polζ, are nearly UV immutable, suggesting severe loss of function. A strain with this substitution in Polα (pol1-Y869A) is viable, but it exhibits slow growth, sensitivity to hydroxyurea, and a spontaneous mutator phenotype for frameshifts and base substitutions. The pol1-Y869A/pol1-Y869A diploid exhibits aberrant growth. Thus, this tyrosine is critical for the function of all four eukaryotic family B DNA polymerases. Strains with a tyrosine substitution in the conserved NS/VxYG motif in region III in Polα, -δ, or -ε are viable and a strain with the homologous substitution in Rev3 is UV mutable. The Polα mutant has no obvious phenotype. The Polε (pol2-Y831A) mutant is slightly sensitive to hydroxyurea and is a semidominant mutator for spontaneous base substitutions and frameshifts. The Polδ mutant (pol3-Y708A) grows slowly, is sensitive to hydroxyurea and methyl methanesulfonate, and is a strong base substitution and frameshift mutator. The pol3-Y708A/pol3-Y708A diploid grows slowly and aberrantly. Mutation rates in the Polα, -δ, and -ε mutant strains are increased in a locus-specific manner by inactivation of PMS1-dependent DNA mismatch repair, suggesting that the mutator effects are due to reduced fidelity of chromosomal DNA replication. This could result directly from relaxed base selectivity of the mutant polymerases due to the amino acid changes in the polymerase active site. In addition, the alanine substitutions may impair catalytic function to allow a different polymerase to compete at the replication fork. This is supported by the observation that the pol3-Y708A mutation is recessive and its mutator effect is partially suppressed by disruption of the REV3 gene.

scholarly journals The effect of attachment site mutations on strand exchange in bacteriophage lambda site-specific recombination.

Genetics ◽  
1989 ◽  
Vol 122 (4) ◽  
pp. 727-736
Author(s):  
C E Bauer ◽  
J F Gardner ◽  
R I Gumport ◽  
R A Weisberg
Keyword(s):  
Attachment Site ◽  
Strand Exchange ◽  
Segregation Pattern ◽  
Base Substitution ◽  
Base Pairs ◽  
Multiple Mutation ◽  
Attachment Sites ◽  
Dna Strands ◽  
Substitution Mutations ◽  
Overlap Region

Abstract Recombination of phage lambda attachment sites occurs by sequential exchange of the DNA strands at two specific locations. The first exchange produces a Holliday structure, and the second resolves it to recombinant products. Heterology for base substitution mutations in the region between the two strand exchange points (the overlap region) reduces recombination; some mutations inhibit the accumulation of Holliday structures, others inhibit their resolution to recombinant products. To see if heterology also alters the location of the strand exchange points, we determined the segregation pattern of three single and one multiple base pair substitution mutations of the overlap region in crosses with wild type sites. The mutations are known to differ in the severity of their recombination defect and in the stage of strand exchange they affect. The three single mutations behaved similarly: each segregated into both products of recombination, and the two products of a single crossover were frequently nonreciprocal in the overlap region. In contrast, the multiple mutation preferentially segregated into one of the two recombinant products, and the two products of a single crossover appeared to be fully reciprocal. The simplest explanation of the segregation pattern of the single mutations is that strand exchanges occur at the normal locations to produce recombinants with mismatched base pairs that are frequently repaired. The segregation pattern of the multiple mutation is consistent with the view that both strand exchanges usually occur to one side of the mutant site. We suggest that the segregation pattern of a particular mutation is determined by which stage of strand exchange it inhibits and by the severity of the inhibition.

scholarly journals A fetal globin gene mutation in A gamma nondeletion hereditary persistence of fetal hemoglobin increases promoter strength in a nonerythroid cell.

1988 ◽  
Vol 8 (2) ◽  
pp. 713-721 ◽  
Author(s):  
M W Rixon ◽  
R E Gelinas
Keyword(s):  
Transient Expression ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Type A ◽  
Base Substitution ◽  
Globin Genes ◽  
Wild Type ◽  
Gamma Globin ◽  
Base Substitutions ◽  
Hereditary Persistence

Single base substitutions have been identified in the promoter regions of A gamma-globin genes from individuals with certain types of nondeletion A gamma hereditary persistence of fetal hemoglobin (HPFH). The presence of these mutations is closely associated with the A gamma HPFH phenotype, but proof that they are the nondeletion HPFH determinants is lacking. To test directly whether these base substitutions can result in an increase in A gamma-globin gene transcription, we studied cosmid clones containing the G gamma- through beta-globin gene regions from individuals with Greek-type (G-to-A base substitution at -117) and Chinese-type (C-to-T base substitution at -196) A gamma HPFH in a transient expression assay. When tested as part of a cosmid clone, the Greek HPFH A gamma-globin gene consistently produced about 1.4 times as much RNA as the wild-type A gamma-globin gene when standardized against RNA transcribed from the G gamma genes in cis. The relative strengths of the normal and HPFH A gamma-globin gene promoters were also compared in transient expression assays with plasmids containing the A gamma-globin genes. Pseudo-wild-type A gamma-globin genes containing a short, transcriptionally neutral deletion were used so that two A gamma-globin genes that differed in their promoter sequences could be compared in the same transfection. The plasmid transient expression results indicated a 1.3- to 1.4-fold increase in steady-state RNA levels from the Greek-type A gamma HPFH promoter compared with the wild-type A gamma promoter, while no difference was documented between the Chinese-type A gamma HPFH promoter and the wild-type A gamma promoter.

Adenovirus origin of DNA replication: sequence requirements for replication in vitro

1987 ◽  
Vol 7 (2) ◽  
pp. 864-874
Author(s):  
R J Wides ◽  
M D Challberg ◽  
D R Rawlins ◽  
T J Kelly
Keyword(s):  
Base Substitution ◽  
Base Pairs ◽  
Viral Origin ◽  
Sequence Organization ◽  
Origin Of Dna Replication ◽  
Sequence Elements ◽  
Specific Nucleotide Sequence ◽  
Origin Function ◽  
Sequence Requirements

The initiation of adenovirus DNA takes place at the termini of the viral genome and requires the presence of specific nucleotide sequence elements. To define the sequence organization of the viral origin, we tested a large number of deletion, insertion, and base substitution mutants for their ability to support initiation and replication in vitro. The data demonstrate that the origin consists of at least three functionally distinct domains, A, B, and C. Domain A (nucleotides 1 to 18) contains the minimal sequence sufficient for origin function. Domains B (nucleotides 19 to 40) and C (nucleotides 41 to 51) contain accessory sequences that significantly increase the activity of the minimal origin. The presence of domain B increases the efficiency of initiation by more than 10-fold in vitro, and the presence of domains B and C increases the efficiency of initiation by more than 30-fold. Mutations that alter the distance between the minimal origin and the accessory domains by one or two base pairs dramatically decrease initiation efficiency. This critical spacing requirement suggests that there are specific interactions between the factors that recognize the two regions.

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