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. 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. Many prokaryotic transcriptional activators bind to a specific DNA sequence at or near the promoter and affect transcription initiation by making direct contacts with RNA polymerase. Several studies have shown that changing the juxtaposition of the activator binding site with respect to the promoter can alter the effectiveness of the activator protein (2Gaston K. Bell A. Kolb A. Buc H. Busby S. Cell. 1990; 62: 733-743Abstract Full Text PDF PubMed Scopus (205) Google Scholar, 3Woody S.T. Fong R.S.-C. Gussin G.N. J. Mol. Biol. 1993; 229: 37-51Crossref PubMed Scopus (25) Google Scholar, 4Joung J.K. Le L.U. Hochschild A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3083-3087Crossref PubMed Scopus (53) Google Scholar, 5Lavigne M. Kolb A. Buc H. Biochemistry. 1992; 31: 9647-9656Crossref PubMed Scopus (24) Google Scholar). Moreover, the ability of certain proteins to activate or repress transcription initiation depends on the sequence to which they bind (6Monsalve M. Mencia M. Rojo F. Salas M. EMBO J. 1996; 15: 383-391Crossref PubMed Scopus (49) Google Scholar, 7Starr D.B. Matsui W. Thomas J.R. Yamamoto K.R. Genes Dev. 1996; 10: 1271-1283Crossref PubMed Scopus (109) Google Scholar). These observations indicate that proper alignment of the activator surface and the RNA polymerase is critical for functional catalysis of transcription initiation by the activator protein. Hence, alterations in the geometry of the ternary complex formed between RNA polymerase, the activator protein, and DNA may be expected to influence the efficiency of transcription initiation. A variety of sequence-dependent DNA structure effects, such as twist and twisting deformability of the DNA, the width of the major and/or minor grooves, the ability of the DNA to bend or sequence-directed intrinsic bends within the DNA, have been shown to influence protein-DNA complex stability (8Koudelka G.B. Harbury P.H. Harrison S.C. Ptashne M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4633-4637Crossref PubMed Scopus (102) Google Scholar, 9Koudelka G.B. Nucleic Acids Res. 1991; 19: 4115-4119Crossref PubMed Scopus (22) Google Scholar, 10Koudelka G.B. Carlson P. Nature. 1992; 355: 89-91Crossref PubMed Scopus (72) Google Scholar, 11Wu L. Koudelka G.B. J. Biol. Chem. 1993; 268: 18975-18981Abstract Full Text PDF PubMed Google Scholar, 12Gartenberg M.R. Crothers D.M. Nature. 1986; 333: 824-829Crossref Scopus (232) Google Scholar). Given that the activation surfaces of the transcriptional activator proteins must be precisely aligned with their specific targets, DNA sequence-dependent differences in the conformation of the activator protein-DNA complexes may alter their efficiency of transcriptional activation. Despite this realization, very little data exist concerning the role of DNA sequence-dependent structure on transcriptional activator function. This is in part the result of the lack of knowledge concerning the effects of variations in binding site sequence on the conformation of protein-DNA complexes. In this paper, we explore the effects of sequence-dependent differences in the conformation of a bacteriophage 434 repressor-DNA complex on its transcriptional activation function. The repressor protein of bacteriophage 434 is a helix-turn-helix-containing DNA-binding protein encoded by thecI gene. This protein controls the developmental fate of the phage by acting as a transcriptional regulator. The bacteriophage chromosome contains two operator regions OR and OL, each of which is divided into three repressor binding sites. The repressor binds to each of six binding sites or operators in the bacteriophage chromosome with differing affinities. This binding site discrimination is critical for the phage's choice between lytic and lysogenic development. For example, in OR, the repressor binds with highest affinity to two sites, OR1 and OR2. In this configuration, the repressor bound at OR2 activates transcription at the PRMpromoter, presumably by contacting RNA polymerase, leading to expression of the genes that are responsible for maintenance of the lysogenic state (13Bushman F.D. Ptashne M. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 9353-9357Crossref PubMed Scopus (26) Google Scholar, 14Bushman F.D. J. Mol. Biol. 1993; 230: 28-40Crossref PubMed Scopus (31) Google Scholar). This binding configuration also permits the repressor to concurrently inhibit transcription of genes needed for lytic phage growth by repressing transcription from the PRpromoter. Higher concentrations of the repressor result in its binding to OR3, causing repression of PRMtranscription. The repressor binds its site as a dimer of identical subunits. The sequences of these sites display incomplete rotational symmetry (for examples, see Fig. 1). The 2-fold related “recognition” α-helices, one from each protein monomer, lie in successive major grooves on one face of the DNA. Each recognition helix is positioned in the major groove so that its side chains can make base-specific contacts with the outermost 4 or 5 base pairs in each half of the operator sequence. The repressor makes no base-specific contacts to the central 4 bases of the site. Earlier work established that sequence-specific differences in DNA structure and/or flexibility of the noncontacted bases at position 6–9 at the center of the 434 operator affect both the affinity of the 434 repressor for operator and modulates the repressor's ability to recognize the base pairs at specific operator positions (1Bell A.C. Koudelka G.B. J. Mol. Biol. 1993; 234: 542-553Crossref PubMed Scopus (34) Google Scholar, 8Koudelka G.B. Harbury P.H. Harrison S.C. Ptashne M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4633-4637Crossref PubMed Scopus (102) Google Scholar, 15Bell A.C. Koudelka G.B. J. Biol. Chem. 1995; 270: 1205-1212Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 16Koudelka G.B. Harrison S.C. Ptashne M. Nature. 1987; 326: 886-888Crossref PubMed Scopus (181) Google Scholar). Noncontacted bases affect the 434 repressor's affinity and specificity for operator through two related structural phenomena. First, sequence-dependent differences in the structure of the unbound DNA and the varying abilities of these structures to be distorted affect overall affinity of operator for the repressor (8Koudelka G.B. Harbury P.H. Harrison S.C. Ptashne M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4633-4637Crossref PubMed Scopus (102) Google Scholar). Second, the sequence of the noncontacted bases affects specificity and affinity by preventing optimal interaction between the repressor's DNA contacting residues and the contacted bases (1Bell A.C. Koudelka G.B. J. Mol. Biol. 1993; 234: 542-553Crossref PubMed Scopus (34) Google Scholar, 15Bell A.C. Koudelka G.B. J. Biol. Chem. 1995; 270: 1205-1212Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). These conformational changes are manifested in an increased nuclease sensitivity of particular DNA sequences in complex with the repressor. The effects of noncontacted bases sequence on operator affinity for the repressor are important in determining the life cycle choices of the phage; the differences between the noncontacted base sequences of OR1, OR2, and OR3 prevent the repressor from recognizing the position 4 base in OR3, but allow recognition of the base at this position in OR1 and OR2 (15Bell A.C. Koudelka G.B. J. Biol. Chem. 1995; 270: 1205-1212Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar)1. The binding of the 434 repressor to OR2 is necessary and sufficient for activation of transcription F.D. J. Mol. Biol. 1993; 230: 28-40Crossref PubMed Scopus (31) Google Scholar). bound at OR2, the repressor RNA polymerase at (13Bushman F.D. Ptashne M. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 9353-9357Crossref PubMed Scopus (26) Google Scholar). the conformation of the 434 repressor-DNA complexes with DNA that the alignment of the activation surface on the repressor may with binding site sequence. We data the that the conformation of the as by sequence-dependent differences in DNA affects the ability of the 434 repressor to activate We also the structural that may be related to sequence-dependent differences in repressor transcriptional activation. and 434 as by P. of 434 DNA Scholar). RNA polymerase from or as H. K. A. 1996; PubMed Google Scholar). polymerase a of the of the from by the H. K. A. 1996; PubMed Google Scholar). and from from with DNA from of the by a and from G. J. Ptashne M. J. Mol. Google by This which 434 OR, and into L. G. Nucleic Acids Res. PubMed Scopus Google that been and The transcription by the from this in OR2, OR3 and/or promoter sequence Fig. for by polymerase as in A. Nucleic Acids Res. 1992; PubMed Scopus Google Scholar, as the polymerase A the promoter from and in with as F.D. J. Mol. Biol. 1993; 230: 28-40Crossref PubMed Scopus (31) Google Scholar). each DNA 5 or with varying of 434 repressor for at in the transcription and for to the of RNA polymerase to a of and for at to allow the of complexes. The transcription by the addition of and and of and of the by addition of and on The of RNA from initiation at to the of by of these as L. Koudelka G.B. J. Biol. Chem. 1993; 268: 18975-18981Abstract Full Text PDF PubMed Google Scholar). a DNA from the the and This or with varying concentrations of 434 repressor at for by the addition of RNA polymerase. varying of at the DNA to for The by and the DNA by two The DNA in of and for at to at the The DNA in the of in and on a The by as in L. A. Koudelka G.B. J. Biol. Chem. 1992; Full Text PDF PubMed Google The DNA as The DNA with of 434 repressor in transcription of at sufficient to on one in 5 of The with and in a and on changes in the DNA sequence bound by the repressor affect the conformation of the 434 complex (1Bell A.C. Koudelka G.B. J. Mol. Biol. 1993; 234: 542-553Crossref PubMed Scopus (34) Google A.C. Koudelka G.B. J. Biol. Chem. 1995; 270: 1205-1212Abstract Full Text Full Text PDF PubMed Scopus (13) Google and G. Koudelka, we that these sequence changes may alter the ability of the repressor to with the RNA polymerase. This by the ability of the repressor to activate transcription bound at and OR OR in the of the from RNA polymerase initiation at the promoter are As shown (13Bushman F.D. Ptashne M. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 9353-9357Crossref PubMed Scopus (26) Google Scholar, 14Bushman F.D. J. Mol. Biol. 1993; 230: 28-40Crossref PubMed Scopus (31) Google of the 434 repressor to the transcription from and transcription from PRM. of at the to that needed to OR1 and OR2 these A and These the transcriptional role of the repressor at and the of the repressor on transcription F.D. J. Mol. Biol. 1993; 230: 28-40Crossref PubMed Scopus (31) Google Scholar, M. A Scholar). As also shown concentrations of the repressor results in repression of results show that repression of results from OR3 by the repressor preventing the binding of RNA polymerase to the of 434 repressor to on and are concentrations increased in 2-fold at 434 OR, the differences between the concentrations of the repressor to that repress are this we a in OR3 that its affinity for the repressor Fig. for Fig. that the repressor activates from this as as from the repressor is needed to to see repression of transcription on this on the As the ability of the repressor to repress transcription is to of the repressor binding to OR3 of the effects of OR2 on transcription from in the studies we the as Fig. and that transcription activation of by the repressor bound at an OR2 an A at position 4 Fig. for is as with that the repressor is bound to OR2. Fig. that the in transcription is the repressor the OR2. Fig. also that the activation is to the of of OR3 by the repressor on these this the repressor to be to bind OR3 at concentrations the activation by the repressor from the OR2 is a result of a DNA of the from the DNA-bound repressor to RNA polymerase and by affecting binding site to the of position 4 affects transcription activation by the repressor bound at OR2, we the of transcription activation by the repressor. studies (1Bell A.C. Koudelka G.B. J. Mol. Biol. 1993; 234: 542-553Crossref PubMed Scopus (34) Google Scholar, 15Bell A.C. Koudelka G.B. J. Biol. Chem. 1995; 270: 1205-1212Abstract Full Text Full Text PDF PubMed Scopus (13) Google that both the conformation and stability of complexes are to we also that position 4 A the of operator affinity for the with changes in the conformation of the complex (15Bell A.C. Koudelka G.B. J. Biol. Chem. 1995; 270: 1205-1212Abstract Full Text Full Text PDF PubMed Scopus (13) Google The data in Fig. 4 show that the of transcription from OR2 is by changing the between and As we from the binding studies (1Bell A.C. Koudelka G.B. J. Mol. Biol. 1993; 234: 542-553Crossref PubMed Scopus (34) Google the of repressor needed to is also by changes in In to the results with the the of transcription of on the an A at position 4 in OR2. Despite the increased sensitivity of the repressor's binding to the OR2, binding show that at or the repressor the OR2 Hence, the of on transcription activation from the position 4 operator its on binding to the repressor. We established that position 4 affects affinity by changing the conformation of the complex (1Bell A.C. Koudelka G.B. J. Mol. Biol. 1993; 234: 542-553Crossref PubMed Scopus (34) Google these position conformational changes may also affect the repressor transcription activation. of binding that the conformation of the complexes position 4 can be to a configuration that the complex by changing the of the base at position (15Bell A.C. Koudelka G.B. J. Biol. Chem. 1995; 270: 1205-1212Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). that position 4 base sequence affects the repressor's ability to activate transcription by altering the conformation of the we the of the base at operator position in position 4 base sequence and the repressor's ability to activate on these Fig. that changing the position base from to or the of the position 4 A on transcriptional activation by the repressor. with the of position on the repressor binding of a position 4 binding site (15Bell A.C. Koudelka G.B. J. Biol. Chem. 1995; 270: 1205-1212Abstract Full Text Full Text PDF PubMed Scopus (13) Google the of position on transcriptional activation by the repressor is This a between the conformation of the complex and the efficiency with which the repressor can activate In with the effects of position on the repressor transcription activation on the position 4 OR2 changing position in the of the position 4 sequence the ability of the repressor to activate In the of a position 4 base changing the base at position from transcriptional activation by changing position to the repressor's ability to activate transcription by The differences in the of position on activation in OR2 with their on the conformation of the the data in that changing the sequence of the binding site of the 434 repressor the ability of the repressor to activate These sequence-dependent effects changing the sequence of contacted or noncontacted DNA structural changes affect transcription activation by the we the of changing one of the residues that operator position to This protein sequence the position 4 base specificity of the repressor G.B. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar). show that changing this the DNA at operator position 4 to to the protein and also changes the of the two DNA-bound S.C. 1990; PubMed Scopus Google Scholar). and S. to the the the ability of the repressor to activate transcription from OR2 by the of changing position 4 from a on transcriptional activation by the repressor on the repressor. The of the DNA on activation by the repressor with the of the operator sequence on binding of this protein. These observations are with the that the position 4 A the repressor's ability to activate transcription by altering the conformation of the A for the of position 4 on transcriptional activation is that the A affects the ability of RNA polymerase to bind the The in OR2 is base pairs from the transcription site of and is in a position to affect interaction of the promoter DNA with the DNA base changes in this of the may affect protein contacts by the of the of RNA polymerase G.N. K. A. J. 1992; PubMed Google Scholar, K. A. J. 1996; PubMed Google Scholar). this we the of OR2 on the ability of the repressor to activate transcription of an RNA polymerase an in which the of this been results show that changing position 4 from A transcription by both RNA polymerase and RNA polymerase the The identical of the position 4 sequence on the of transcription by these two that the transcription is to effects on the protein DNA of the into the position 4 sequence changes affect the repressor's ability to activate transcription an of the repressor The repressor by RNA polymerase to the or may transcription by the of an RNA complex to between these we the sequence of the of and the ability of RNA polymerase to these in the and of the repressor Fig. for of the sequence of that this promoter sequences in both and the sequence of the of the ability of RNA polymerase to this DNA in the of the repressor the regions of and changing the sequence of changes the sequence of from As expected from this sequence Fig. A also that the the ability of RNA polymerase to transcription at The repressor is to transcription on the the and the of transcription indicate that repressor binding is by the in PRM. These with the that changing the sequence of the of the promoter no on transcription initiation in the of repressor are with the that the of transcription from may be to between RNA polymerase and the of the In the the repressor may to or these protein-DNA contacts The that the repressor is to activate transcription from the promoter that the repressor activates transcription by RNA polymerase to PRM. This by the of complex in with the for the of in the of the repressor on or OR2. Fig. that the of complex as by with on both and These are by in the of formed of these data that the at which both complexes and are formed are and by the sequence of OR2. the OR2 the repressor's ability to a RNA polymerase that complex we that the of complex and be This is Moreover, the that the of transcription the of complex on both the and that the OR2 is affecting in the of RNA polymerase from the to The results in Fig. A and show that both the of formed and of complexes detected on the OR2 are with the OR2 This is with a affinity of RNA polymerase for the as with the These results suggest that the OR2 A the ability of the repressor to RNA polymerase contacts with the promoter and RNA polymerase Moreover, the that the overall of is to a by OR2 is the of complexes formed is also with this the of RNA polymerase promoter complexes the overall through the complex the of complexes be by RNA polymerase binding affinity is The data presented here indicate that the ability of a protein to activate transcription depends the sequence of its DNA binding site. the sequences of contacted or noncontacted bases in the binding site of the 434 repressor its ability to activate transcription from PRM. These DNA sequence changes to affect transcription by altering the conformation of the protein-DNA shown that the conformation of a protein-DNA complex can its ability to activate we to the in conformation of the from the 434 repressor to RNA polymerase. we can we must the of the activation by the repressor. for the can be In the the repressor may transcription from by the of a particular DNA the DNA and its may the the repressor may transcription by with RNA polymerase. the conformation of the complex may the of this interaction by the accessibility of the surface of the repressor that is responsible for with RNA polymerase. lines of evidence suggest that the repressor transcription a direct protein with RNA polymerase. First, F.D. Ptashne M. Cell. 1988; Full Text PDF PubMed Scopus Google in the 434 repressor that affect DNA but are in transcriptional activation. the DNA binding and transcriptional activation of the repressor are with the that the repressor activates transcription by making a with RNA polymerase. with structural studies A. M. Ptashne M. Harrison S.C. 1988; PubMed Scopus Google Scholar, Harrison S.C. J. Mol. Biol. 1993; PubMed Scopus Google the results of and indicate that these repressor lie in a that be in direct with the of RNA polymerase F.D. J. Mol. Biol. 1993; 230: 28-40Crossref PubMed Scopus (31) Google Scholar, F.D. Harrison S.C. Ptashne M. Nature. PubMed Scopus Google Scholar). Second, Fig. 5 that identical position sequence changes in two position 4 have effects on transcriptional activation by DNA-bound 434 repressor. is to that sequence changes affect DNA structure in precisely on the sequence at position Hence, the 434 repressor to activate transcription by making a direct protein with RNA polymerase. changes in the conformation of the complex to with transcriptional activation by the repressor by altering the geometry of the interaction between the repressor and RNA polymerase. that the complex structure modulates the interaction between the repressor and RNA polymerase permits to DNA sequence changes to the transcriptional work that changing the sequence of bases at operator positions 4 and/or can result in an of the position of the protein with respect to the DNA (15Bell A.C. Koudelka G.B. J. Biol. Chem. 1995; 270: 1205-1212Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). these structural alterations affect the 434 polymerase is that the in the position of the repressor with respect to the DNA RNA polymerase with the surface of the repressor. We this by the between a in the surface of the repressor and the DNA that is to be contacted by both RNA polymerase and the repressor in the ternary transcription We the structures of the 434 repressor and its bound to DNA binding sites A. M. Ptashne M. Harrison S.C. 1988; PubMed Scopus Google Scholar, Harrison S.C. J. Mol. Biol. 1993; PubMed Scopus Google Scholar, Harrison S.C. 1993; Full Text PDF PubMed Scopus Google with to the between the in part responsible for transcription activation by the 434 and the that is to the position These show that this is 4 in the complex Harrison S.C. 1993; Full Text PDF PubMed Scopus Google is in complexes between repressor and operators a base at position 4 Harrison S.C. J. Mol. Biol. 1993; PubMed Scopus Google The between these two is also in complexes between the operator and the repressor the and S. These observations suggest that the structural by the protein and DNA sequence alterations the repressor's ability to activate transcription by preventing RNA polymerase from the surface of the repressor. The structural for the transcription data an in the activation of the 434 repressor that is from prokaryotic activators such as S. Cell. Full Text PDF PubMed Scopus Google Scholar). As the in the 434 repressor that its ability to activate transcription are in the helix of its This structural is also responsible for DNA binding by the repressor. one of the for the activation is to be in the of the of RNA polymerase that contacts the of the promoter Hochschild A. J. Google Scholar, M. H. PubMed Scopus Google Scholar, H. J. Mol. Biol. PubMed Scopus Google Scholar, Cell. 1992; Full Text PDF PubMed Scopus Google Scholar). The by of the DNA binding regions with in both repressor and RNA polymerase, and the for DNA binding by that changes in the geometry of the repressor's interaction with operator and/or the of the two repressor on DNA affect the geometry of the The alterations in conformation to affect transcription activation by preventing the repressor from the RNA the repressor is to transcription by RNA polymerase bound at the promoter a this that the complex may transcription from by the affinity of RNA polymerase for the show that the repressor transcription from by the of an that is RNA polymerase J. Mol. Biol. PubMed Scopus Google Scholar, Cell. Full Text PDF PubMed Scopus Google Scholar). in the two proteins lie at positions F.D. Ptashne M. Cell. 1988; Full Text PDF PubMed Scopus Google Scholar, A. Ptashne M. Cell. Full Text PDF PubMed Scopus Google and the structure of the two complexes are S.C. 1990; PubMed Scopus Google Scholar, A. M. Ptashne M. Harrison S.C. 1988; PubMed Scopus Google Scholar, 1992; PubMed Scopus Google Scholar, 1988; PubMed Scopus Google is to their differences in activation may this The the that the of an promoter complex is a The of each and in the to an complex is by a that with promoter sequence. For the RNA that are in with the protein and DNA may with sequence. Hence, the of between two identical of promoter complexes may be or of RNA polymerase this and 434 repressor the chemical but this is as an on the affinity of RNA polymerase for the promoter or of a bound promoter complex may with promoter sequence. The is on the that the between the that is by both RNA polymerase and repressor binding sites and the surface of the repressor that contacts RNA polymerase between and 434 complexes Harrison S.C. J. Mol. Biol. 1993; PubMed Scopus Google Scholar, 1988; PubMed Scopus Google Scholar). This that the of the regions of the on the RNA polymerase may be a is repressor transcriptional activators have on the A. B. K. A. J. Mol. Biol. PubMed Scopus Google Scholar). The differences between the activation surface on the RNA polymerase by the two proteins may to the differences in their activation The results we here sequence differences between binding sites for a particular activator protein to its activation function. is established that sequence differences allow an activator protein to between binding sites a of DNA and that this discrimination is for proper of the The data presented here show that in addition to binding binding site sequence can alter the activation efficiency of the protein.