Genetic studies of the lac repressor

Kleina, Lynn G.; Miller, Jeffrey H

doi:10.1016/0022-2836(90)90126-7

Cited by 172 publications

(89 citation statements)

References 62 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…2). The effects observed for 2AP or P nucleotides base paired with uracil at these positions are entirely consistent with this interpretation ( (42,43). In general, the wild-type interaction has demonstrated asymmetry mutations and substitutions in the left half of the operator always result in greater changes in affinity than those in the right half (9,14) and crosslinking experiments underscore the differences between the two halves (44).…”

Section: Direct Interaction Of Major Groove Sites In the Operatormentioning

confidence: 53%

Modified nucleotides reveal the indirect role of the central base pairs in stabilizing thelacrepressor-operator complex

Zhang

Gottlieb

1995

Nucl Acids Res

View full text Add to dashboard Cite

Guanine residues in the lac operator were replaced by 2-aminopurine or purine analogues, pairing the modified nucleotides with C. The observed equilibrium dissociation constants for lac repressor binding to substituted operators were measured in 10 mM Tris, 150 mM KCI, 0.1 mM EDTA, 0.1 mM DTE, pH 7.6 at 250C. These measurements revealed five positions that destabilized the complex when substituted with either analogue. Two positions, which are related by a 2-fold symmetry, are in the major groove of the operator thought to directly interact with the protein. Three sites were in the central region of the operator. A purine analogue at a sixth site perturbed the local DNA structure and destabilized the complex. Alkylation interference experiments of the 2-aminopurine substituted operators demonstrated that, of the five affected, two substitutions displayed altered phosphate interference patterns at the phosphate adjacent to the substituted base. For these operators, complex formation was measured in different concentrations of KCI to assess the contribution of counterion release to the bimolecular process. The results indicated that both complexes were similar to wild-type, although minor changes were observed. The Iobs of the complex was then measured when 2-aminopurine or purine analogues were paired with uracil nucleotide, a base pair that serves to stabilize the DNA. The introduction of the new base pairs revealed two effects on the bimolecular interaction. For those operator sites that are thought to perturb the interaction directly, the affinity of the complex was weakened to levels observed for the singly-substituted operators. In contrast, the nucleotides of 2-aminopurine paired with uracil positioned in the central region of the operator served to enhance the stability of the complex. The purine-uracil base pair substitution op the other hand had a significant destabilizing effect on the interaction. We propose that the central base pairs modulate binding of the complex by altering the intrinsic properties of the DNA. Two specific attributes are required to achieve the lowest free energy of interaction. The DNA must have two interstrand hydrogen bonds to stabilize the duplex and it must have properties associated with directional bending or unwinding. This analysis does not rule out contributions by direct interactions between the protein and the central region of the operator but underscores how indirect effects play a major role in complex formation in this system.

show abstract

Section: Direct Interaction Of Major Groove Sites In the Operatormentioning

confidence: 53%

Modified nucleotides reveal the indirect role of the central base pairs in stabilizing thelacrepressor-operator complex

Zhang

Gottlieb

1995

Nucl Acids Res

View full text Add to dashboard Cite

show abstract

“…The E. coli suppressor strains were kindly provided by Dr. Jeffrey Miller (UCLA) (24). In addition, the tRNA suppressor plasmids for Gly, Ala, Cys, Lys, Glu/Gln, 5 Arg, Pro, His, and Phe, were transferred into the genetic background of E. coli BL21(DE3).…”

Section: Methodsmentioning

confidence: 99%

“…On the other hand, transformation of the non-His-tagged g17 plasmid, which expresses a more active gp17 than the His-tagged gp17, results in lethality (12, 28). (24). The effect of each aa substitution was assessed by plaque forming ability of Y253am on the corresponding suppressor strain.…”

Section: B Aspmentioning

confidence: 99%

See 1 more Smart Citation

Functional Analysis of the Bacteriophage T4 DNA-packaging ATPase Motor

Mitchell¹,

Rao

2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

Packaging of double-stranded DNA into bacteriophage capsids is driven by one of the most powerful force-generating motors reported to date. The phage T4 motor is constituted by gene product 16 (gp16) (18 kDa; small terminase), gp17 (70 kDa; large terminase), and gp20 (61 kDa; dodecameric portal). Extensive sequence alignments revealed that numerous phage and viral large terminases encode a common Walker-B motif in the N-terminal ATPase domain. The gp17 motif consists of a highly conserved aspartate (Asp 255 ) preceded by four hydrophobic residues ( 251 MIYI 254 ), which are predicted to form a ␤-strand. Combinatorial mutagenesis demonstrated that mutations that compromised hydrophobicity, or integrity of the ␤-strand, resulted in a null phenotype, whereas certain changes in hydrophobicity resulted in cs/ts phenotypes. No substitutions, including a highly conservative glutamate, are tolerated at the conserved aspartate. Biochemical analyses revealed that the Asp 255 mutants showed no detectable in vitro DNA packaging activity. The purified D255E, D255N, D255T, D255V, and D255E/ E256D mutant proteins exhibited defective ATP binding and very low or no gp16-stimulated ATPase activity. The nuclease activity of gp17 is, however, retained, albeit at a greatly reduced level. These data define the N-terminal ATPase center in terminases and show for the first time that subtle defects in the ATP-Mg complex formation at this center lead to a profound loss of phage DNA packaging.In double-stranded DNA bacteriophages and herpes viruses, genome packaging is the fulfillment of viral DNA metabolism and an indispensable step in the assembly of infectious virions. Packaging is initiated by the endonucleolytic cleavage of the newly synthesized concatemeric DNA, which in T4 is a highly branched, head-to-tail polymeric network with very few, if any, ends (1, 2). The cleavage is catalyzed by holoterminase, a nonstructural, multisubunit complex, composed of the small subunit gp16 3 (18 kDa) and the large subunit gp17 (70 kDa) (3). The cleaved end is linked to the unique dodecameric portal vertex of the empty prohead through specific interactions between the terminase and the portal protein (gp20, 61 kDa). A packaging motor is thus assembled, which drives directional translocation of DNA into the prohead by an ATP-dependent mechanism (4). Following head filling, the terminase 4 makes a second cut, and the terminase-DNA complex disassociates from the packaged head and reassociates with another empty prohead to continue head filling in a processive fashion.Numerous DNA packaging models have been proposed, yet the basic mechanism is still a mystery (5-7). Single molecule packaging studies determined that the phage DNA-packaging motor is the strongest molecular motor measured to date (4). Cryoelectron microscopy imaging and atomic structure of phage Phi29 portal are consistent with the symmetry mismatch model, which postulates that the mismatch between the 5-fold viral capsid and 12-fold portal allows an ATP-driven portal rotation that is c...

show abstract

“…Therefore, in this study, we analyzed 83 of the 557 residues in KdpA to identify those that are critical for substrate binding. To test comprehensively the importance of each residue, we examined at least 11 substitutions at each residue using the amber suppression technique (8,9). This technique creates up to 12 amino acid substitutions at an introduced amber stop codon.…”

mentioning

confidence: 99%

Substrate-binding Clusters of the K+-transporting Kdp ATPase of Escherichia coli Investigated by Amber Suppression Scanning Mutagenesis

Dorus¹,

Mimura²,

Epstein

2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

The Kdp-ATPase of Escherichia coli is a four-subunit P-type ATPase that accumulates K ؉ with high affinity and specificity. ؉ binding by this pump has been analyzed in detail by site-directed mutagenesis. We have examined 83 of the 557 residues in KdpA, from 11 to 34 residues in each of four binding clusters known to affect K ؉ binding. Amber mutations were constructed in a plasmid carrying the kdpFABC structural genes. Transferring these plasmids to 12 suppressor strains, each inserting a different amino acid at amber codons, created 12 different substitutions at the mutated sites. This study delineates the four clusters and confirms that they are important for K ؉ affinity but have little effect on the rate of transport. At only 21 of the residues studied did at least three substitutions alter affinity for K ؉ , an indication that a residue is in or very near a K ؉ binding site. At many residues lysine was the only substitution that altered its affinity. The effect of lysine is most likely a repulsive effect of this cationic residue on K ؉ and thus reflects the effective distance between a residue and the site of binding or passage of K ؉ in KdpA. Once a crystallographic structure of Kdp is available, this measure of effective distance will help identify the path of K ؉ as it moves through the KdpA subunit to cross the membrane.

show abstract

Genetic studies of the lac repressor

Cited by 172 publications

References 62 publications

Modified nucleotides reveal the indirect role of the central base pairs in stabilizing thelacrepressor-operator complex

Modified nucleotides reveal the indirect role of the central base pairs in stabilizing thelacrepressor-operator complex

Functional Analysis of the Bacteriophage T4 DNA-packaging ATPase Motor

Substrate-binding Clusters of the K+-transporting Kdp ATPase of Escherichia coli Investigated by Amber Suppression Scanning Mutagenesis

Contact Info

Product

Resources

About