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S D Goodman

Publications and source records attributed to S D Goodman.

18 recordsLinked to original sources
PubMed2000

DNA bending and twisting properties of integration host factor determined by DNA cyclization.

The binding of many proteins to DNA is profoundly affected by DNA bending, twisting, and supercoiling. When protein binding alters DNA conformation, interaction between inherent and induced DNA conformation can affect protein binding affinity and specificity. Integration host factor (IHF), a sequence-specific, DNA-binding protein of Escherichia coli, strongly bends the DNA upon binding. To assess the influence of inherent DNA bending on IHF binding, we took advantage of the high degree of natural static curvature associated with an IHF site on a 163-bp minicircle and measured the binding affinity of IHF for its recognition site contained on this DNA in both circular and linear form. IHF showed a higher affinity for the circular form of the DNA when compared to the linear form. In addition, the presence of IHF during DNA cyclization changed the topology of cyclization products and their ability to bind IHF, consistent with IHF untwisting DNA. These results show that inherent DNA conformation anisotropy is an important determinant of IHF binding affinity and suggests a mechanism for modulation of IHF activity by local DNA conformation.

Bacterial Proteins↗
PubMed2000

Characterization of the gtfB and gtfC promoters from Streptococcus mutans GS-5.

We have characterized the promoter regions of the gtfB and gtfC genes from Streptococcus mutans GS-5. Using a plasmid-based reporter system, we discovered that the gtfC promoter is an order of magnitude stronger than the gtfB promoter. In addition, we found that a variety of growth conditions failed to affect or discriminate between the expression of the two promoters. Only during the various phases of growth could we demonstrate significant changes in expression. We conclude that gtfB and gtfC promoters are coordinately expressed.

Base Sequence↗
PubMed1999

Replacement of integration host factor protein-induced DNA bending by flexible regions of DNA.

The Escherichia coli integration host factor (IHF) protein is required for site-specific recombination of bacteriophage lambda DNA. Previously, we had shown that alternative modules of static DNA curvature could partially replace IHF in recombination. Now we use regions of single-stranded DNA as a flexible tether to address whether the function of IHF in recombination is simply to reduce persistence length. Although we find that these modules clearly enhance recombination in the absence of IHF, they are not perfect replacements. In addition, evidence is presented that the efficacy of a flexibility swap is specific to a particular IHF site. This may indicate that additional functions beyond simple deformation of DNA are required of IHF. During the course of these experiments we discovered that these flexible sequences are still specific sites for IHF binding and function.

Bacterial Proteins↗
PubMed1999

Global structure similarities of intact and nicked DNA complexed with IHF measured in solution by fluorescence resonance energy transfer.

We have analyzed the structure of two related protein-DNA complexes consisting of integration host factor (IHF) bound to two different versions of the H' site of bacteriophage lambda. Both DNA substrates were 55 bp in length. While one was native duplex the other possessed a nick in one strand at a crucial position within the IHF consensus at the same position as in the reported crystal structure of the DNA-IHF complex. By labeling the 5'-ends of these DNA molecules with donor and acceptor fluorescent dyes, we were able to measure the distance between the dyes by fluorescence resonance energy transfer (FRET) and model DNA distortion. The FRET efficiency decreased from 0.49 +/- 0.01 (nicked DNA) to 0.37 +/- 0.01 (intact DNA) when the gap in the DNA strand was closed. The measured dye-to-dye distance of IHF in complex with nicked DNA was in agreement with the expected value from the crystal structure. Although we found that the two structures were distinguishable, the global shape induced by IHF was retained between the two DNA molecules. Furthermore, our FRET and modeling techniques have sufficiently high resolution to distinguish subtle changes in nucleoprotein complexes with biological relevance.

Bacterial Proteins↗
PubMed1999

Firefly luciferase as a reporter to study gene expression in Streptococcus mutans.

The utility of firefly luciferase as a reporter was tested in Streptococcus mutans. Under control of an endogenous promoter, the luciferase coding sequence was strongly expressed, while a promoterless version was indistinguishable from the background. Luciferase activity was easily extracted and the assay rapid and reproducible. In addition, the half-life of luciferase activity was found to be comparable to those of other frequently used reporters. Thus, firefly luciferase can readily be used as a reporter in S. mutans, a useful alternative to methods requiring radioactive isotopes.

Animals↗
PubMed1999

Growth phase-dependent regulation of nuoA-N expression in Escherichia coli K-12 by the Fis protein: upstream binding sites and bioenergetic significance.

The expression of the nuoA-N operon of Escherichia coli K-12, which encodes the proton-pumping NADH dehydrogenase I is modulated by growth phase-dependent regulation. Under respiratory growth conditions, expression was stimulated in early exponential, and to a lesser extent in late exponential and stationary growth phases. The stimulation in the early exponential growth phase was not observed in fis mutants, which are deficient for the growth phase-responsive regulator Fis. Neither the alternative sigma factor RpoS nor the integration host factor (IHF) are involved in growth phase-dependent regulation of this operon. When incubated with nuo promoter DNA, isolated Fis protein formed three retarded complexes in gel mobility experiments. DNase I footprinting identified three distinct binding sites for Fis, 237 bp (fis1), 197 bp (fis2) and 139 bp (fis3) upstream of the start of the major transcript of nuoA-N, T1. The protein concentrations required for half-maximal binding to fis1, fis2 and fis3 were about 20 nM, 40 nM and 100 nM Fis, respectively. The IHF protein bound 82 bp upstream of the start of transcript T2 with a half-maximal concentration for binding of 50 nM. Due to the growth phase-dependent regulation by Fis, the synthesis of the coupling NADH dehydrogenase I is increased relative to that of the noncoupling NADH dehydrogenase II during early exponential growth. This ensures higher ATP yields under conditions where large amounts of ATP are required.

Bacterial Proteins↗
PubMed1999

In vitro selection of integration host factor binding sites.

Integration host factor (IHF) is a bacterial protein that binds and severely bends a specific DNA target. IHF binding sites are approximately 30 to 35 bp long and are apparently divided into two domains. While the 3' domain is conserved, the 5' domain is degenerate but is typically AT rich. As a result of physical constraints that IHF must impose on DNA in order to bind, it is believed that this 5' domain must possess structural characteristics conducive for both binding and bending with little regard for specific contacts between the protein and the DNA. We have examined the sequence requirements of the 5' binding domain of the IHF binding target. Using a SELEX procedure, we randomized and selected variants of a natural IHF site. We then analyzed these variants to determine how the 5' binding domain affects the structure, affinity, and function of an IHF-DNA complex in a native system. Despite finding individual sequences that varied over 100-fold in affinity for IHF, we found no apparent correlation between affinity and function.

Bacterial Proteins↗
PubMed1997

Knowledge-based model of a glucosyltransferase from the oral bacterial group of mutans streptococci.

Mutans streptococci glucosyltransferases catalyze glucosyl transfer from sucrose to a glucan chain. We previously identified an aspartyl residue that participates in stabilizing the glucosyl transition state. The sequence surrounding the aspartate was found to have substantial sequence similarity with members of alpha-amylase family. Because little is known of the protein structure beyond the amino acid sequence, we used a knowledge-based interactive algorithm, MACAW, which provided significant level of homology with alpha-amylases and glucosyltransferase from Streptococcus downei gtfI (GTF). The significance of GTF similarity is underlined by GTF/alpha-amylase residues conserved in all but one alpha-amylase invariant residues. Site-directed mutagenesis of the three GTF catalytic residues are homologous with the alpha-amylase catalytic triad. The glucosyltransferases are members of the 4/7-superfamily that have a (beta/alpha)8-barrel structure and belong to family 13 of the glycohydralases.

Algorithms↗
PubMed1996

The complete nucleotide sequence of bacteriophage HP1 DNA.

The complete nucleotide sequence of the temperate phage HP1 of Haemophilus influenzae was determined. The phage contains a linear, double-stranded genome of 32 355 nt with cohesive termini. Statistical methods were used to identify 41 probable protein coding segments organized into five plausible transcriptional units. Regions encoding proteins involved in recombination, replication, transcriptional control, host cell lysis and phage production were identified. The sizes of proteins in the mature HP1 particle were determined to assist in identifying genes for structural proteins. Similarities between HP1 coding sequences and those in databases, as well as similar gene organizations and control mechanisms, suggest that HP1 is a member of the P2-like phage family, with strong similarities to coliphages P2 and 186 and some similarity to the retronphage Ec67.

Bacteriophage P2↗
PubMed1994

Architectural elements in nucleoprotein complexes: interchangeability of specific and non-specific DNA binding proteins.

Integration host factor (IHF) is required in lambda site-specific recombination to deform the DNA substrates into conformations active for recombination. HU, a homolog of IHF, can also deform DNA but binds without any apparent sequence specificity. We demonstrate that HU can replace IHF by cooperating with the recombinase protein, integrase, to generate a stable and specific complex with electrophoretic mobility and biochemical activity very close to the complex formed by IHF and integrase. The eukaryotic HMG1 and HMG2 proteins differ entirely in structure from HU but they also bind DNA non-specifically and induce or stabilize deformed DNA. We show that the eukaryotic HMG1 and HMG2 proteins cooperate with integrase at least as well as does HU to make a defined structure. We also find that the eukaryotic core histone dimer H2A-H2B can replace IHF, suggesting that the histone dimer is functional outside the context of a nucleosome. HU and the HMG proteins not only contribute to the formation of stable complexes, but they can at least partially replace IHF for the integrative and excisive recombination reactions. These results, together with our analysis of nucleoprotein complexes made with damaged recombination sites, lead us to conclude that the cooperation between HU and integrase does not depend on protein-protein contacts. Rather, cooperation is manifested through building of higher order structures and depends on the capacity of the non-specific DNA binding proteins to bend DNA. While all these non-specific binding proteins appear to fulfil the same bending function, they do so with different efficiencies. This probably reflects subtle structural differences between the assembled complexes.

Allosteric Regulation↗
PubMed1992

Deformation of DNA during site-specific recombination of bacteriophage lambda: replacement of IHF protein by HU protein or sequence-directed bends.

Escherichia coli IHF protein is a prominent component of bacteriophage lambda integration and excision that binds specifically to DNA. We find that the homologous protein HU, a nonspecific DNA binding protein, can substitute for IHF during excisive recombination of a plasmid containing the prophage attachment sites attL and attR but not during integrative recombination between attP and attB. We have examined whether IHF and HU function in excisive recombination is mediated through DNA bending. Our strategy has been to construct chimeric attachment sites in which IHF binding sites are replaced by an alternative source of DNA deformation. Previously, we demonstrated that properly phased bends can substitute for the binding of IHF at one site in attP. Although this result is highly suggestive of a critical role of IHF-promoted bending in lambda integration, its interpretation is obscured by the continued need for IHF binding to the remaining IHF sites of these constructs. In the present work, we engineered a population of sequence-directed bends in the vicinity of the two essential IHF sites found in attR and attL. Even in the absence of IHF or HU, pairs of these attachment sites with properly phased bends are active for both in vitro and in vivo excision. This success, although tempered by the limited efficiency of these systems, reinforces our interpretation that IHF functions primarily as an architectural element.

Attachment Sites, Microbiological↗
PubMed1991

Factors influencing the specific interaction of Neisseria gonorrhoeae with transforming DNA.

The specific interaction of transformable Neisseria gonorrhoeae with DNA depends on the recognition of specific 10-residue target sequences. The relative affinity for DNA between 3 and 17 kb in size appears to be linearly related to the frequency of targets on the segment and is unaffected by absolute size. The average frequency of targets in chromosomal DNA of N. gonorrhoeae appears to be approximately one per 1,000 bp.

DNA, Bacterial↗
PubMed1989

Functional replacement of a protein-induced bend in a DNA recombination site.

In recent years the capacity of proteins to bend DNA by binding to specific sites has become a widely appreciated phenomenon. In many cases, the protein-DNA interaction is known to be functionally significant because destruction of the DNA site or the protein itself results in an altered phenotype. An important question to be answered in these cases is whether bending of DNA is important per se or is merely a consequence of the way a particular protein binds to DNA. Here we report direct evidence from the bacteriophage lambda integration system that a bend introduced by a protein is intrinsically important. We find that a binding site for a specific recombination protein known to bend DNA can be successfully replaced by two other modules that also bend DNA; related modules that fail to bend DNA are ineffective.

Bacterial Proteins↗
PubMed1989

Nucleotide sequence and expression of the gene for the site-specific integration protein from bacteriophage HP1 of Haemophilus influenzae.

The nucleotide sequence of the leftmost 2,363 base pairs of the HP1 genome, which includes the attachment site (attP) and the integration region, was determined. This sequence contained an open reading frame encoding a 337-residue polypeptide, which is a member of the integrase family of site-specific recombination proteins as judged by sequence comparison. The open reading frame was located immediately adjacent to the att site and was oriented so that initiation of translation would begin distal to the att site and end in its immediate vicinity. Expression of this DNA segment in Escherichia coli provided extracts which promoted site-specific recombination between plasmids containing cloned HP1 attP and Haemophilus influenzae attB sites. This recombination was directional, since no reaction was observed between plasmids containing attR and attL sites. The reaction was stimulated by the accessory protein integration host factor of E. coli. Evidence was also obtained that the integration host factor influenced the levels of HP1 integrase expression. The deduced amino acid sequence of HP1 integrase has remarkable similarity to that deduced for the integrase of coliphage 186.

Amino Acid Sequence↗
PubMed1988

Identification and arrangement of the DNA sequence recognized in specific transformation of Neisseria gonorrhoeae.

DNA segments from Neisseria gonorrhoeae, cloned and propagated in Escherichia coli, were tested for the ability to competitively inhibit gonococcal transformation. The nucleotide sequences of active segments were determined and compared; these sequences contained the sequence 5' GCCGTCTGAA 3' in common. Subcloning studies confirmed the identity of this sequence as the gonococcal DNA recognition site. The three instances of the recognition sequence isolated from N. gonorrhoeae chromosomal DNA contain the sequence in the immediate neighborhood of its inverted repeat. Because a single copy of the sequence functions as a recognition site, the inverted duplication is not required for specific binding. The dyad symmetric arrangements of the chromosomal recognition sequences may form stable stem-loop structures that can function as terminators or attenuators of transcription. These inverted repeats are located at the boundaries of long open reading frames. The recognition sequence also constitutes part of two other probable terminators of gonococcal genes. We conclude that the signal for recognition of transforming DNA by gonococci is a frequent component of transcriptional terminator sequences. This regulatory function might account for the origin and maintenance of recognition sequences in the chromosomes of Gram-negative transformable bacteria.

Base Sequence↗
PubMed1987

Nucleotide sequences and properties of the sites involved in lysogenic insertion of the bacteriophage HP1c1 genome into the Haemophilus influenzae chromosome.

Bacteriophage HP1c1 lysogenizes its host Haemophilus influenzae Rd by inserting its genome into the bacterial chromosome. The DNA segments corresponding to the integration regions on the phage and host chromosomes and the two junctions formed between phage and host sequences on lysogenic insertion were isolated and propagated in Escherichia coli HB101 as hybrid plasmids by using pBR322 as the vector. The nucleotide sequences in the vicinity of the point of recombinational insertion were determined. Phage and host DNA shared an extensive, nearly identical, segment that was 183 base pairs long. This segment consisted of 93 identical residues and a 27-residue portion containing 6 mismatches, followed by 63 identical residues. Recombinational insertion occurred within the 63-residue identical segment and involved neither duplication nor deletion of any residues. Short inverted repeats consisting of clustered A-T base pairs were present within the two 27-residue segments. Two additional sites on the host chromosome showed significant hybridization to the phage-host homology region.

Bacteriophages↗