Biomedical subjects

G B Koudelka

Publications and source records attributed to G B Koudelka.

At least 19 recordsLinked to original sources

PubMed2001

Repression of transcription initiation at 434 P(R) by 434 repressor: effects on transition of a closed to an open promoter complex.

The lambdoid bacteriophage repressors function both as transcription activators and repressors. Regulation of transcription at the adjacent, but divergent promoters, P(RM) and P(R), determines the phage's choice between the lytic and lysogenic development pathways. Here, we demonstrate that 434 repressor bound at 434 O(R)1 alone is not sufficient to repress transcription from 434 P(R,) but that 434 repressor bound at 434 O(R)2 alone is necessary and sufficient to repress P(R )transcription. This is different from what occurs in the related bacteriophage lambda, in which binding of lambda repressor to either lambdaO(R)1 or lambdaO(R)2 represses transcription from lambdaP(R). The combined results of gel mobility shift and KMnO(4) footprinting assays show that while 434 repressor binding to 434 O(R)2 does not preclude RNA polymerase binding at the P(R) promoter, it does prevent it from forming open complexes at this promoter. The RNA polymerase-P(R) complexes that form in the presence of repressor are heparin-resistant and the DNA is not melted. This observation indicates that 434 repressor bound at 434 O(R)2 inhibits transcription initiation at the P(R) promoter by "locking" the RNA polymerase-P(R) complex into an inactive state instead of "blocking" the access of RNA polymerase to promoter DNA.

Bacteriophage lambda↗

PubMed2001

Function-based selection and characterization of base-pair polymorphisms in a promoter of Escherichia coli RNA polymerase-sigma(70).

We performed two sets of in vitro selections to dissect the role of the -10 base sequence in determining the rate and efficiency with which Escherichia coli RNA polymerase-sigma(70) forms stable complexes with a promoter. We identified sequences that (i) rapidly form heparin-resistant complexes with RNA polymerase or (ii) form heparin-resistant complexes at very low RNA polymerase concentrations. The sequences selected under the two conditions differ from each other and from the consensus -10 sequence. The selected promoters have the expected enhanced binding and kinetic properties and are functionally better than the consensus promoter sequence in directing RNA synthesis in vitro. Detailed analysis of the selected promoter functions shows that each step in this multistep pathway may have different sequence requirements, meaning that the sequence of a strong promoter does not contain the optimal sequence for each step but instead is a compromise sequence that allows all steps to proceed with minimal constraint.

Base Sequence↗

PubMed2000

Cooperativity: action at a distance in a classic system.

A new high resolution crystal structure of the phage lambda repressor reveals the basis for repressor dimer formation and, together with biochemical data, provides insights into the mechanism of repressor tetramer formation, a process essential to the cooperative binding and gene regulatory activities of this protein.

DNA↗

PubMed2000

DNA sequence requirements for the activation of 434 P(RM) transcription by 434 repressor.

A dimer of the 434 repressor bound at O(R)2 activated transcription initiation from P(RM) by contacting RNA polymerase. Although DNA-binding site mutations at either end of O(R)2 decreased the ability of the repressor to activate P(RM) transcription, mutations proximal to the promoter had a greater effect on transcription activation. Orienting a repressor subunit bearing the altered specificity Gln-28 --> Ala mutation to the halfsite of O(R)2 proximal to the P(RM) promoter decreased the repressor's ability to activate transcription initiation at 434 P(RM) to a much greater extent than if this subunit was placed in the O(R)2 half-site distal to P(RM). In addition to showing that the downstream (promoter proximal) subunit of the O(R)2-bound 434 repressor functions in activating 434 P(RM), the results indicated that DNA sequence-dependent conformational changes alter the efficiency with which the repressor activates P(RM) transcription. These unexpected findings highlight the importance of the structure of the repressor-DNA interface in activating transcription from P(RM).

Amino Acid Substitution↗

PubMed2000

Mutually exclusive utilization of P(R) and P(RM) promoters in bacteriophage 434 O(R).

Establishment and maintenance of a lysogen of the lambdoid bacteriophage 434 require that the 434 repressor both activate transcription from the P(RM) promoter and repress transcription from the divergent P(R) promoter. Several lines of evidence indicate that the 434 repressor activates initiation of P(RM) transcription by occupying a binding site adjacent to the P(RM) promoter and directly contacting RNA polymerase. The overlapping architecture of the P(RM) and P(R) promoters suggests that an RNA polymerase bound at P(R) may repress P(RM) transcription initiation. Hence, part of the stimulatory effect of the 434 repressor may be relief of interference between RNA polymerase binding to the P(RM) promoter and to the P(R) promoter. Consistent with this proposal, we show that the repressor cannot activate P(RM) transcription if RNA polymerase binds at P(R) prior to addition of the 434 repressor. However, unlike the findings with the related lambda phage, formation of RNA polymerase promoter complexes at P(RM) and at P(R) apparently are mutually exclusive. We find that the RNA polymerase-mediated inhibition of repressor-stimulated P(RM) transcription requires the presence of an open complex at P(R). Taken together, these results indicate that establishment of an open complex at P(R) directly prevents formation of an RNA polymerase-P(RM) complex.

Bacteriophage lambda↗

PubMed1999

DNA-induced conformational changes in bacteriophage 434 repressor.

Although bacteriophage 434 repressor binds to its specific DNA sites only as a dimer, formation of the dimers in solution occurs at concentrations three orders of magnitude higher than those needed to bind the 434 operator DNA. Our results suggest that both specific and non-specific DNA induce conformational changes in repressor that lead to formation of repressor dimers. The repressor conformational changes induced by DNA occur at concentrations much lower than those needed for binding of repressor, suggesting that the alternative conformations of repressor persist even if the protein is not in direct contact with DNA. Hence, DNA acts in a "catalytic" fashion to induce a steady-state amount of an alternative repressor conformation that has an enhanced affinity for its specific binding site. These findings suggest that the repressor conformer induced by non-specific DNA is the form of the repressor that is optimized for searching for DNA binding sites along non-specific DNA. Upon finding a binding site, the repressor protein undergoes an additional conformational change that allows it to "lock-on" to its specific site.

Acrylamide↗

PubMed1998

Carboxyl-terminal domain dimer interface mutant 434 repressors have altered dimerization and DNA binding specificities.

Strong dimerization of the repressor, mediated by the carboxyl (C)-terminal domain, is a prerequisite for forming a specific complex with DNA and cooperative DNA binding to form tetramers. We have generated a computer model of the C-terminal domain of the 434 repressor based on the crystal structure of the homologous UmuD' protein. This model predicts that residues in the primary sequence between 93 and 168 contribute to the dimer interface. We changed several amino acid residues located in this region. Gel filtration and crosslinking assays were used to characterize the strength and specificity of dimerization of the purified repressor C-terminal domain dimer interface mutants. These results indicate that amino acid residues K121, H139, D161 and N163 contribute to the strength and/or specificity of dimerization. The relative affinity of the bacteriophage 434 repressor for 434 operators is determined, in part, by the repressor's ability to detect sequence-dependent structural alterations in the non-contacted region at the center of an operator site. We find that the relative ability of C-terminal domain dimer interface mutant repressors to dimerize does not necessarily predict their relative abilities to bind DNA, and that these proteins are deficient in detecting non-contacted base-dependent differences in operator strength. Our results show that the structure of the DNA in complex with these mutant proteins differs from that found in wild-type repressor-operator complexes, even though the sites of these mutations lie in a separate domain from that which contacts the DNA. These observations demonstrate that the structural integrity of the C-terminal domain dimer interface is required to appropriately orient the DNA binding information contained within the DNA-contacting N-terminal domain.

Amino Acid Sequence↗

PubMed1998

DNA-based positive control mutants in the binding site sequence of 434 repressor.

As detected by chemical nuclease treatments, the conformation of the 434 repressor-DNA complex depends on the sequence of the bound DNA (Bell, A. C., and Koudelka, G. B. (1993) J. Mol. Biol. 234, 542-553). We show here that these DNA sequence-dependent conformational changes alter the efficiency with which the repressor activates transcription from 434 PRM. Several lines of evidence suggest that binding site sequence affects the repressor's ability to activate transcription by altering the accessibility of the activation surface on the repressor to RNA polymerase. The results presented here show that in addition to affecting transcription by altering the overall binding affinity of protein for DNA, DNA sequence may also modulate the activity of the DNA-bound protein.

Base Sequence↗

PubMed1998

Recognition of DNA structure by 434 repressor.

In complexes of bacteriophage 434 binding sites with 434 repressor the central 4 bp of the 14 bp site are not contacted by the protein, although changes in these bases alter binding site affinity for the repressor. Our previous data suggested that the ability of the non-contacted central bases to be overtwisted in repressor-DNA complexes governs affinity of the binding site for 434 repressor. This idea was tested by examining the affinity of two central sequence variant 434 binding sites for 434 repressor as a function of binding site average twist. The 434 repressor preferred the relatively overwound binding site to the two more underwound forms. The greatest affinity enhancement resulting from increasing twist was observed with a binding site that is relatively underwound and more resistant to twisting deformation. Consistent with the idea that 434 repressor overtwists its binding site upon DNA binding, we show that 434 repressor is capable of binding to sites bearing a single base insertion in their center (a 15mer), but binds poorly to binding sites bearing central base deletions (12mer and 13mer). The N-terminal dimer interface plays a large role in determining 434 repressor central base preferences. Mutations in this interface eliminate central base discrimination and/or site size preferences. These mutations also lead to changes in the size of the repressor footprint on the various sized DNA sites that are consistent with their binding characteristics.

Amino Acid Sequence↗

PubMed1997

Recognition of nonconserved bases in the P22 operator by P22 repressor requires specific interactions between repressor and conserved bases.

The ability of P22 repressor protein to distinguish between the six naturally occurring operator binding sites is critically important in determining whether the bacteriophage chooses to grow lytically or lysogenically. We have shown that changes in the highly conserved bases at P22 operator positions 3, 5, 6, and 7 prevent specific binding of P22 repressor. Moreover, studies of mutant proteins identified the three repressor amino acids that directly contact these conserved bases. The pattern of operator sequence conservation permits these direct amino acid-base pair interactions to occur in all except one of the 12 operator half-sites in the phage chromosome. Therefore, repressor differential affinity for these sites cannot be due to these highly conserved base pair-amino acid interactions. Our binding studies show that the nonconserved bases at positions 2 and 4 also play an important role in determining the relative affinity of the naturally occurring P22 operators for P22 repressor. Our data indicate that the direct contacts between the three solvent-exposed amino acids and the conserved bases in the binding site lock these amino acids in place, forming a scaffold allowing the rest of the amino acids side chains to form weaker interactions with the nonconserved bases in the binding site.

Amino Acid Sequence↗

PubMed1997

DNA-based loss of specificity mutations. Effects of DNA sequence on the contacted and non-contacted base preferences of bacteriophage P22 repressor.

Although the two central bases of the P22 operator are not contacted by the P22 repressor, changes in these bases alter the affinity of operator for repressor. Previous studies (Wu, L., and Koudelka, G. B. (1993) J. Biol. Chem. 268, 18975-18981) show that the structure of the P22 repressor-operator complex varies with central base sequence. Here we show that central base sequence composition affects the strength of two, and likely all, specific amino acid-base pair contacts between synthetic P22 operators and P22 repressor. However, altering a specific protein-DNA contact via a loss-of-contact mutation in repressor results in a loss of specificity at only one contacted position. Thus, only changing the sequence of non-contacted bases affects repressor's global base specificity. The observed effects of ionic concentration on the affinities of various operators for repressor and the DNase I patterns of protein complexes with these binding sites indicate certain central base sequences facilitate optimal juxtaposition of repressor with its contacted bases, while others prevent it. The existence of different structural forms of the repressor-operator complexes explains how the relative energetic importance of specific amino acid-base pair edge contacts is modulated.

DNA↗

PubMed1997

Dimerization specificity of P22 and 434 repressors is determined by multiple polypeptide segments.

The repressor protein of bacteriophage P22 binds to DNA as a homodimer. This dimerization is absolutely required for DNA binding. Dimerization is mediated by interactions between amino acids in the carboxyl (C)-terminal domain. We have constructed a plasmid, p22CT-1, which directs the overproduction of just the C-terminal domain of the P22 repressor (P22CT-1). Addition of P22CT-1 to DNA-bound P22 repressor causes the dissociation of the complex. Cross-linking experiments show that P22CT-1 forms specific heterodimers with the intact P22 repressor protein, indicating that inhibition of P22 repressor DNA binding by P22CT-1 is mediated by the formation of DNA binding-inactive P22 repressor:P22CT-1 heterodimers. We have taken advantage of the highly conserved amino acid sequences within the C-terminal domains of the P22 and 434 repressors and have created chimeric proteins to help identify amino acid regions required for dimerization specificity. Our results indicate that the dimerization specificity region of these proteins is concentrated in three segments of amino acid sequence that are spread across the C-terminal domain of each of the two phage repressors. We also show that the set of amino acids that forms the cooperativity interface of the P22 repressor may be distinct from those that form its dimer interface. Furthermore, cooperativity studies of the wild-type and chimeric proteins suggest that the location of cooperativity interface in the 434 repressor may also be distinct from that of its dimerization interface. Interestingly, changes in the dimer interface decreases the ability of the 434 repressor to discriminate between its wild-type binding sites, O(R)1, O(R)2, and O(R)3. Since 434 repressor discrimination between these sites depends in large part on the ability of this protein to recognize sequence-specific differences in DNA structure and flexibility, this result indicates that the C-terminal domain is intimately involved in the recognition of sequence-dependent differences in DNA structure and flexibility.

Amino Acid Sequence↗

PubMed1995

How 434 repressor discriminates between OR1 and OR3. The influence of contacted and noncontacted base pairs.

The sequence of the bacteriophage 434 OR1 (ACAAAACTTTCTTGT) differs from its OR3 (ACAGTTTTCTTGT) at positions 4-6. X-ray analysis shows that the side chain of Gln33 of the 434 repressor makes van der Waals' and H-bond contacts with the T at position 4' in complex with OR1, but no specific contact is observed at this position in 434 repressor-OR3 complexes. No contacts are made by repressor to the bases at positions 5 or 6 in either binding site. The significance of the sequence differences between OR1 and OR3 in determining the operator affinity for repressor were examined by constructing synthetic variants of these operators. Measurements of the affinity of these operators for repressor as a function of ionic strength revealed that although base pairs 5 and 6 are not contacted by 434 repressor, they can nonetheless influence operator affinity for repressor by modulating the degree to which ionic interactions contribute to the overall binding energy. Both the magnitude and direction of their effect depends on the status of repressor's contacts to the bases at position 4. The role of contact made by Gln33 to position 4 was examined by mutating this amino acid to Ala and by examining the affinity of wild type repressor for an operator bearing a 5-methylcytosine at position 4' in an OR1-4G mutant. These experiments showed that repressor's preferences at operator positions 5 and 6 are linked to its position 4 preference via a van der Waals' contact between amino acid 33 and a methyl group on the base at operator position 4'. Together, the results of the experiments shown here reveal that bases that do not contact the protein alter its preferences for bases at the contacted operator position 4.

Amino Acids↗

PubMed1994

Expression, purification, and functional characterization of the carboxyl-terminal domain fragment of bacteriophage 434 repressor.

The repressor protein of bacteriophage 434 binds to DNA as a dimer of identical subunits. Its strong dimerization is mediated by the carboxyl-terminal domain. Cooperative interactions between the C-terminal domains of two repressor dimers bound at adjacent sites can stabilize protein-DNA complexes formed with low-affinity binding sites. We have constructed a plasmid, pCT1, which directs the overproduction of the carboxyl-terminal domain of 434 repressor. The protein encoded by this plasmid is called CT-1. Cells transformed with pCT1 are unable to be lysogenized by wild-type 434 phage, whereas control cells are lysogenized at an efficiency of 1 to 5%. The CT-1-mediated interference with lysogen formation presumably results from formation of heteromeric complexes between the phage-encoded repressor and the plasmid-encoded carboxyl-terminal domain fragment. These heteromers are unable to bind DNA and thereby inhibit the repressor's activity in promoting lysogen formation. Two lines of evidence support this conclusion. First, DNase I footprinting experiments show that at a 2:1 ratio of CT-1 to intact 434 repressor, purified CT-1 protein prevents the formation of complexes between 434 repressor and its OR1 binding site. Second, cross-linking experiments reveal that only a specific heterodimeric complex forms between CT-1 and intact 434 repressor. This latter observation indicates that CT-1 interferes with 434 repressor-operator complex formation by preventing dimerization and not by altering the conformation of the DNA-bound repressor dimer. Our other evidence is also consistent with this suggestion. We have used deletion analysis in an attempt to define the region which mediates the 434 repressor-CT-1 interaction. CT-1 proteins which have more than the last 14 amino acids removed are unable to interfere with 434 repressor action in vivo.

Amino Acid Sequence↗

PubMed1993

Operator sequence context influences amino acid-base-pair interactions in 434 repressor-operator complexes.

The 434 repressor binds more tightly to OR1 than it does to OR3. The repressor makes several specific contacts with the symmetrically arrayed outer four base-pairs of the 14 base-pair site, and no specific contacts to the central six base-pairs. The sequence of the outer base-pairs of OR1 and OR3 differs only by an A-->G substitution at position 4 in one half-site of OR3, while that of central bases is very different. As expected from sequence analysis of wild-type operators, the data show repressor prefers an A.T base-pair at position 4. The magnitude of this preference depends on operator sequence context and solution conditions. Position 4 changes in the context of OR1 have a greater effect on operator affinity for 434 repressor than do similar changes in OR3. Although OR1 and OR3 display different affinities for 434 repressor, their repressor-operator complexes are similarly insensitive to changes in salt concentration and temperature. By contrast, complexes formed between repressor and position 4 mutant OR1, bearing an A.T-->G.C change, and OR3, which bears a G.C-->A.T change, are affected greatly, and to similar extents, by changes in ionic strength and temperature. Nuclease protection experiments show that 434 repressor protects the DNA phosphate backbone of wild-type operators from cleavage more efficiently than those of mutant operators. These data show that the biochemical and structural properties of a repressor-operator complex, while affected by position 4 base sequence, are independent of the identity of this base. The ability of repressor to recognize the base at position 4 depends on the sequence context at operator positions 5 to 7. Apparently there is an interplay between the bases at operator positions 4 to 7 which has a global effect on the structure of the repressor-operator complex.

Base Composition↗

PubMed1993

Differential recognition of OR1 and OR3 by bacteriophage 434 repressor and Cro.

The developmental decisions of bacteriophage 434 depend on the ability of 434 repressor and Cro to bind OR1 and OR3 with different relative affinities; repressor binds OR1 tighter than OR3, whereas Cro slightly prefers OR3 over OR1. Studies with operator mutants show that repressor's lower relative affinity for OR3 results from a deviation in the sequence of OR3 from consensus; an A-->G change at position 4 in one half-site (OR1: A-C-A-A-A-C-T-T-T-C-T-T-G-T; OR3: A-C-A-G-T-T-T-T-T-C-T-T-G-T). Similar experiments show that Cro binds operators containing either A.T or G.C bases pairs at position 4 equally well, but cannot bind operators containing C.G or T.A base pairs at this position. A Gln33-->Ala mutation in 434 repressor diminishes, but does not eliminate, its ability to distinguish between purines at position 4. This shows that a glutamine at amino acid 33 is not the sole determinant of repressor's position 4 specificity. Changing Gln33-->Leu, the amino acid at the homologous position in Cro, does not confer "Cro-like" position 4 base specificity on repressor. Similarly, a Cro protein bearing Gln at this position does not exhibit repressor's position 4 base preferences. The residual specificities of these mutant proteins indicates that in each protein, more than 1 amino acid is responsible for recognizing bases at position 4. These were identified by analyzing the binding specificities of multiply mutated repressors, in vitro. The types of substitutions made were guided by sequence homologies between 434 repressor and Cro. At least three mutations are needed to eliminate repressor's position 4 base specificity; Gln33-->Ala, Glu32-->Gln, and Thr27-->Lys, although no set of amino acid substitutions in repressor was able to confer Cro-like position 4 specificity to repressor. These results indicate that at least the amino acids at these positions are involved in recognition of the position 4 base. Other evidence suggests that Cro and repressor use identical amino acids present at homologous positions in the DNA recognition helix in different ways.

Amino Acids↗

PubMed1993

Sequence-dependent differences in DNA structure influence the affinity of P22 operator for P22 repressor.

Although the central bases of the P22 operator are not contacted by P22 repressor, the affinity of repressor for operator varies with the sequence of these bases. The KMnO4 and .OH radical susceptibilities of the central bases of the operator vary with their sequence, whether or not the operator is complexed with protein or free in solution. These data show that the minor groove of a lower affinity operator which bears central C-G bases (9C) is more open than that of the higher affinity 9T operator, which bears central T-A bases. This difference in minor groove width is seen both in the absence and presence of repressor. Results of ring closure studies show that, in the absence of repressor, an operator bearing central C.G base pairs operator is overtwisted relative to an operator which contains central T.A base pairs. The binding of P22 repressor unwinds the two operators to similar extents, thereby preserving the relative differences in twists of these DNAs. Although repressor alters the twist of the operator DNA, our results show that differences in DNA torsional flexibility have no role in determining the affinity of operator for protein. Instead, the results indicate that central sequences affect operator affinity for protein by limiting the degree to which the operator can be deformed in the protein-DNA complex. The stability of the complex is apparently modulated, in a central sequence-dependent manner, by alterations in the number and/or geometry of protein-DNA contacts.

Bacteriophage P22↗

PubMed1992

Non-contacted bases affect the affinity of synthetic P22 operators for P22 repressor.

The affinity of synthetic P22 operators for P22 repressor varies with the base sequence at the operator's center. At 100 mM KCl, the affinity of these operators for P22 repressor varies over a 10-fold range. Dimethylsulfate protection experiments indicate that the central bases of the P22 operator are not contacted by the repressor. The KD for the complex of P22 repressor with an operator bearing central T-A bases (9T) increases less than 2-fold between 50 and 200 mM KCl, whereas the KD for the complex of repressor with an operator bearing central C-G bases (9C) increases 10-fold in the same salt range. The DNase I cleavage patterns of both bound and unbound P22 operators also vary with central base sequence. The DNase I pattern of the repressor-9C operator complex changes markedly with salt concentration, whereas that of the 9T operator-repressor complex does not. These changes in nuclease digestion pattern thereby mirror the salt-dependent changes in the P22 operator's affinity for repressor. P22 repressor protects the central base pair of the 9T operator from cleavage by the intercalative cleavage reagent Cu(I)-phenanthroline, while repressor does not protect the central bases of the 9C operator. Together these data indicate that central base pairs affect P22 operator strength by altering the structure of the unbound operator and the repressor-operator complex.

Amino Acid Sequence↗