Flexible Zinc Finger Requirement for Binding of the Transcriptional Activator Staf to U6 Small Nuclear RNA and tRNASec Promoters

The transactivator Staf, which contains seven zinc finger motifs, exerts its effect on gene expression by binding to specific targets in small nuclear RNA (snRNA) and snRNA-type gene promoters. In this work, binding site selection allowed us to identify the 21-base pair ATTACCCATAATGCATYGCGG sequence as the high affinity consensus binding site for Staf. It shows a high sequence divergence with Staf-responsive elements in the Xenopus selenocysteine tRNA (tRNASec) and human U6 snRNA promoters. By using a combination of approaches, we analyzed the interaction of wild-type and truncated Staf zinc finger domains with the consensus, Xenopus tRNASec, and human U6 sites. Two main conclusions emerged from our data. First, the data clearly indicate that zinc finger 7 does not establish base-specific contacts in Staf-DNA complexes. The second conclusion concerns zinc finger 1, which is required for the binding to the Xenopus tRNASec site but is dispensable in the case of the human U6 site. Taking into account the sequence differences in the two sites, these findings demonstrate that Staf utilizes zinc finger 1 in a rather flexible manner, illustrating how a protein can interact with DNAs containing targets of different sequences. The transactivator Staf, which contains seven zinc finger motifs, exerts its effect on gene expression by binding to specific targets in small nuclear RNA (snRNA) and snRNA-type gene promoters. In this work, binding site selection allowed us to identify the 21-base pair ATTACCCATAATGCATYGCGG sequence as the high affinity consensus binding site for Staf. It shows a high sequence divergence with Staf-responsive elements in the Xenopus selenocysteine tRNA (tRNASec) and human U6 snRNA promoters. By using a combination of approaches, we analyzed the interaction of wild-type and truncated Staf zinc finger domains with the consensus, Xenopus tRNASec, and human U6 sites. Two main conclusions emerged from our data. First, the data clearly indicate that zinc finger 7 does not establish base-specific contacts in Staf-DNA complexes. The second conclusion concerns zinc finger 1, which is required for the binding to the Xenopus tRNASec site but is dispensable in the case of the human U6 site. Taking into account the sequence differences in the two sites, these findings demonstrate that Staf utilizes zinc finger 1 in a rather flexible manner, illustrating how a protein can interact with DNAs containing targets of different sequences. selenocysteine tRNA small nuclear RNA Xenopus tRNASec human U6 snRNA base pair glutathione S-transferase zinc finger polymerase chain reaction Regulation of gene expression is mediated by trans-acting proteins that recognize and bind at specific DNA elements in the regulatory regions of the genes. A common feature of many activating proteins is that DNA binding and activation functions reside in different domains of the protein. The majority of sequence-specific DNA-binding proteins can be classified according to the presence of conserved domains, such as helix-turn-helix, zinc finger, or leucine zipper (for reviews, see Refs. 1Pabo C.O. Sauer R.T. Annu. Rev. Biochem. 1992; 61: 1053-1095Crossref PubMed Scopus (1231) Google Scholar and 2Patikoglou G. Burley S.K. Annu. Rev. Biophys. Biomol. Struct. 1997; 26: 289-325Crossref PubMed Scopus (115) Google Scholar). Recently, we have demonstrated that the zinc finger protein Staf, originally identified in Xenopus laevis as the transcriptional activator of the tRNASec 1 gene (3Myslinski E. Krol A. Carbon P. Nucleic Acids Res. 1992; 20: 203-209Crossref PubMed Scopus (50) Google Scholar, 4Schuster C. Myslinski E. Krol A. Carbon P. EMBO J. 1995; 14: 3777-3787Crossref PubMed Scopus (69) Google Scholar), is also involved in transcriptional activation of snRNA and snRNA-type genes, some of which are transcribed by RNA polymerase II and others by RNA polymerase III (5Schaub M. Myslinski E. Schuster C. Krol A. Carbon P. EMBO J. 1997; 16: 173-181Crossref PubMed Scopus (90) Google Scholar). In addition, Staf possesses the capacity to stimulate expression from an RNA polymerase II mRNA promoter. The presence of two physically and functionally distinct activation domains, one specifically activating snRNA-type promoters and the other mRNA promoters, constitutes the molecular basis for this dichotomous transcriptional activity (6Schuster C. Krol A. Carbon P. Mol. Cell. Biol. 1998; 18: 2650-2658Crossref PubMed Scopus (33) Google Scholar). In human cells, ZNF76 and ZNF143 are two human homologs of Staf. ZNF143 is the ortholog, whereas ZNF76 is a DNA-binding protein related to Staf and ZNF143 (7Myslinski E. Krol A. Carbon P. J. Biol. Chem. 1998; 273: 21998-22006Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). In its central part, Staf contains seven contiguous zinc fingers of the C2-H2 type (2Patikoglou G. Burley S.K. Annu. Rev. Biophys. Biomol. Struct. 1997; 26: 289-325Crossref PubMed Scopus (115) Google Scholar, 8Miller J. McLachlan A.D. Klug A. EMBO J. 1985; 4: 1609-1614Crossref PubMed Scopus (1705) Google Scholar). The first six are of the C X4C X3(F/Y)X5L X2H X3H type (X stands for any amino acid), except that the leucine residue is not found in the fourth and fifth fingers, where it is replaced by arginine and tyrosine residues, respectively. The seventh zinc finger, however, is of the C X2C X3Y X5L X2H X4H type. The sequence linking the last histidine of one zinc finger to the first cysteine of the next one is highly conserved, giving rise to the consensus TG(E/D)(K/R)PYN, but the (E/D)(K/R) sequence is not found between the sixth and seventh finger. Comparative DNase I footprinting analysis performed either with the entire protein or with the zinc finger domain only established that the seven tandemly repeated zinc fingers contain the DNA binding domain of Staf (4Schuster C. Myslinski E. Krol A. Carbon P. EMBO J. 1995; 14: 3777-3787Crossref PubMed Scopus (69) Google Scholar). Structural analysis of DNA-zinc finger complexes has demonstrated that each interacting zinc finger contacts 3–5 bp of DNA (9Pavletich N.P. Pabo C.O. Science. 1991; 252: 809-817Crossref PubMed Scopus (1763) Google Scholar, 10Fairall R. Schwabe J.W.R. Chapman L. Finch J.T. Rhodes D. Nature. 1993; 366: 483-487Crossref PubMed Scopus (300) Google Scholar, 11Pavletich N.P. Pabo C.O. Science. 1993; 261: 1701-1707Crossref PubMed Scopus (613) Google Scholar, 12Elrod-Erickson M. Rould M.A. Nekludova L. Pabo C.O. Structure. 1996; 4: 1171-1180Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar, 13Houbaviy H.B. Usheva A. Shenk T. Burley S.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13577-13582Crossref PubMed Scopus (163) Google Scholar, 14Omichinski J.G. Pedone P.V. Felsenfeld G. Gronenborn A.M. Clore G.M. Nat. Struct. Biol. 1997; 4: 122-132Crossref PubMed Scopus (174) Google Scholar, 15Wuttke D.S. Foster M.P. Case D.A. Gottesfeld J.M. Wright P.E. J. Mol. Biol. 1997; 273: 183-206Crossref PubMed Scopus (166) Google Scholar, 16Nolte R.T. Conlin R.M. Harrison S.C. Brown R.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2938-2943Crossref PubMed Scopus (199) Google Scholar). Assuming that all of the seven Staf zinc fingers would contact the DNA, the protein should recognize a binding site composed of at least 21 bp. However, sequence comparisons of known Staf binding sites with the consensus sequence derived from binding site selection revealed a high degree of sequence divergence. This is well illustrated by the Staf-responsive elements that lack the 5′ part of the consensus sequence in the Xenopus tRNASec and the 3′ part in the human U6 promoters (5Schaub M. Myslinski E. Schuster C. Krol A. Carbon P. EMBO J. 1997; 16: 173-181Crossref PubMed Scopus (90) Google Scholar). To elucidate the mechanism by which Staf can recognize divergent DNA sequences, we have assayed the relative contributions of individual zinc fingers, and sets of zinc fingers, to the binding to an optimal site and Staf-responsive elements that are sequence divergent. In this study, determinations of the relative binding affinities of Staf and recombinant zinc finger polypeptides to the optimal site, Xenopus tRNASec, and human U6 Staf-responsive elements in combination with DNase I and binding site This revealed that zinc finger 7 does not establish base-specific contacts with the DNA and that zinc finger 1 is by Staf in a flexible on the site with which it to in (4Schuster C. Myslinski E. Krol A. Carbon P. EMBO J. 1995; 14: 3777-3787Crossref PubMed Scopus (69) Google Scholar). by into the I and sites of the Staf zinc finger domain by of using 5′ and 3′ to and of the Staf respectively. The 3′ contains a The Staf binding site by into the of a DNA containing the optimal Staf binding site. The sequence of the optimal site is The wild-type and truncated Staf zinc finger domains using the glutathione S-transferase gene and by into the and sites of PubMed Scopus Google Scholar), of the zinc finger containing amino and (4Schuster C. Myslinski E. Krol A. Carbon P. EMBO J. 1995; 14: 3777-3787Crossref PubMed Scopus (69) Google Scholar). The in and of the wild-type and truncated zinc finger domains to performed at The proteins using as in PubMed Scopus Google using the with as a site selection performed as in M. Myslinski E. Schuster C. Krol A. Carbon P. EMBO J. 1997; 16: 173-181Crossref PubMed Scopus (90) Google Scholar with the of binding and by performed with protein for the and sixth to DNase I footprinting performed as in 4Schuster C. Myslinski E. Krol A. Carbon P. EMBO J. 1995; 14: 3777-3787Crossref PubMed Scopus (69) Google as by and PubMed Scopus Google Scholar), with of DNA to a of of of at of to the The DNA and in of The in a of in the presence of the DNA and the of protein required to of the The as 1 for at and a containing and from the and analyzed in a a and reaction of the as The as The and of human U6 to and Xenopus tRNASec to by of the using and respectively. The and of the optimal by of the Staf binding site, using and The and to and of respectively. to the relative affinities of the binding sites. In these the protein for at with and of the binding site as the The optimal to a of with for at to establish the The proteins in these by in with the with the and and of the for the in the to and respectively. the and the of binding at each as the of to with A to the for the of binding at known DNA The as the of DNA of the of the a of using a of wild-type or truncated Staf zinc finger domain to glutathione S-transferase of 1 of and of and of the binding reaction the at the and by a The of the in the and DNAs with to on the and with a The can be in of protein to a to that of the analyzed as a of DNA DNA using the from which using binding site identified the sequence as the consensus for of the zinc finger domain of Staf (5Schaub M. Myslinski E. Schuster C. Krol A. Carbon P. EMBO J. 1997; 16: 173-181Crossref PubMed Scopus (90) Google Scholar). this is and and are highly at and In to identify the base for the proteins of glutathione S-transferase to the Staf zinc finger domain in a binding site selection from a containing an of of binding performed with protein for the and sixth to the 21 a with to base with the consensus identified in our work, the derived consensus sequence ATTACCCATAATGCATYGCGG is by an A residue on the 5′ and a residue on the 3′ The that be from this is as A high is for at and at and and a at A for at and at and a at and a C at a for at and for at 1 and and for at and of the data revealed the that identified as binding sites for Staf in the laevis tRNASec and human U6 snRNA promoters (3Myslinski E. Krol A. Carbon P. Nucleic Acids Res. 1992; 20: 203-209Crossref PubMed Scopus (50) Google Scholar, 4Schuster C. Myslinski E. Krol A. Carbon P. EMBO J. 1995; 14: 3777-3787Crossref PubMed Scopus (69) Google Scholar, M. Myslinski E. Schuster C. Krol A. Carbon P. EMBO J. 1997; 16: 173-181Crossref PubMed Scopus (90) Google not in the selection a we that the sites with a affinity with the this using with different the and sites, and the sequence the that we the optimal site It shows to the Staf consensus binding site. The relative of of the two sites and the optimal site to for binding to Staf and Staf zinc finger domain with a of the optimal site It is that the protein of Staf and and Staf zinc finger domain not comparisons of the between and C. that the and sites are by Staf with a affinity that of the optimal site, but with a of the binding Staf and the optimal sequence the and sites, C shows that the Staf zinc finger domain possesses a and binding capacity to the optimal with to the and sites. these indicate that the in the binding site selection highly in the selection of sites that are with a affinity the Staf-responsive elements in the Xenopus tRNASec and human U6 promoters. The amino sequence of the seven zinc fingers of the Staf DNA binding domain is conserved between the Xenopus Staf and its human (7Myslinski E. Krol A. Carbon P. J. Biol. Chem. 1998; 273: 21998-22006Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). In the sequence of the Staf-responsive elements in the Xenopus tRNASec and human U6 promoters are clearly only This the of the of zinc fingers contacts with DNA regulatory that are divergent in sequences. DNase I footprinting a to differences in the of binding of proteins can be using this on DNA derived from to in the laevis tRNASec and to in the human U6 promoters. to the Staf we a of recombinant polypeptides containing the glutathione S-transferase to zinc fingers and from by affinity The DNase I on the Xenopus tRNASec are in A and with a of the data in C. the from to on the and to on the of finger in the of the that of to and on the and respectively. the is from to and the from to of the seventh zinc finger in to a on the with the with of zinc finger 1 in to a the from and on the and respectively. of the and the relative of the protein and Staf the DNA with the the and the amino the of the Staf-responsive I analysis of Staf zinc finger polypeptides binding to the promoter. DNA from base to on the or and with DNase I in the of proteins and and or with of the zinc finger and A and and A and and as and 1 and the tRNASec are of the DNase I The of the and of the Xenopus tRNASec are from to The Staf-responsive is The of DNase I in complexes with and is as and In to the with the Xenopus tRNASec to a on the human U6 at or and however, to DNase I as for the of the human U6 in with a of the data in the a between and the of zinc finger 1 in not the whereas it an of on the of the Xenopus tRNASec that a protein can on two different Staf-responsive and the with and on the human U6 that zinc finger 1 a contact with the U6 Staf-responsive or does not bind the DNA at This with the established with the Xenopus tRNASec in which zinc finger 1 contacts the It is well known that DNase I sequence of it is that not all the a by a protein in a are involved in the the of DNase I can to the all these DNase I footprinting does not an of complexes. in the Staf-responsive elements that are by the wild-type and truncated Staf DNA binding To this using PubMed Scopus Google Scholar). This of the in a DNA The of of the wild-type and truncated zinc finger domains to bind the DNA assayed by in which the Staf complexes from the the and DNAs and analyzed on a for the of the in the and In such an a that is to the a or on the in the containing the DNA that to the protein. a high in the where the DNA This to the contacts by and to of the and optimal sites. The of DNA from these is in and C for the and sites, and a of the data is in in the consensus sequence stands as a the base of the sites to at the of the of the revealed the relative of the individual to the or of a Staf-DNA interacting on of the DNA, contacts to the to be to the between and the sites revealed of any from and on the or on the in the site A and and on the and on the in the optimal site and with the binding of This is different from that with the site, where the is in the 5′ part of the Staf and in its 3′ part and at to and 1 to of the and C and the on or to the and The of in the presence of zinc finger 7 that zinc finger 7 does not establish base-specific contacts with the To this we next analyzed the of by using to binding sites from an of sequences. from the sixth selection and in the protein containing zinc fingers to a consensus to that with the entire finger domain see also these demonstrate that zinc finger 7 does not specifically contact the in Staf-DNA complexes. To the of the zinc finger 1 to the of Staf-DNA we the contacts by to of the and sites. the The for the binding of to the and optimal sites different in 3′ from found for the of zinc finger 1 in a of the of and on the of the and optimal sites, and on the a of the of 7 21 see and for the and optimal sites, and This a of contacts at the of the sites from the zinc finger and that the binding site for zinc finger 1 between base for the site and for the optimal site. In the not the to the site C and in of zinc finger 1 the DNA in the and optimal sites, but not in the site. base-specific contacts are to be for the binding of zinc finger 1 to the DNA, the binding sites with from a of should not contain the zinc finger 1 binding site. The from the sixth of and 7 a of the for each at the 21 It also shows that the containing a consensus of sequence which of the consensus with the entire zinc finger domain at the of each different from that with In addition, the sequence of the consensus not on the that the domain of Staf the of the site, the of this binding selection a highly to our that zinc finger 1 base-specific contacts with the this well with the which that in the base of the optimal site are required for next to the affinities of wild-type and truncated zinc finger domains and for the Xenopus tRNASec, human and optimal sites. This by a of protein with of sites. complexes from the DNA by on a The relative of and DNAs at each DNA and the by analysis of binding In DNA of this the is not by in protein and by the of in the protein Klug A. Nucleic Acids Res. 1993; PubMed Scopus Google Scholar). comparisons between different polypeptides can be found that the affinity of for the and sites is with of and of zinc finger 7 in to an in the for the and sites A different with the of zinc finger 1 in which in and in the but not the for the site. data a to the and site selection which that zinc finger 1 does not establish DNA contacts with the site. the we the for the a of the data that a Staf containing six zinc fingers, either from the amino or has a high binding I also shows that the for the binding of and to the and sites at least of the for the zinc finger and of Staf zinc finger from the of determinations in The by DNA with the and using the and site site from Myslinski site from Myslinski from the of determinations in The by DNA with the and The using the and from Myslinski (7Myslinski E. Krol A. Carbon P. J. Biol. Chem. 1998; 273: 21998-22006Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). in a The zinc finger transcriptional activator Staf to specific in the sequence elements of snRNA and snRNA-type promoters and gene this interaction (3Myslinski E. Krol A. Carbon P. Nucleic Acids Res. 1992; 20: 203-209Crossref PubMed Scopus (50) Google Scholar, 4Schuster C. Myslinski E. Krol A. Carbon P. EMBO J. 1995; 14: 3777-3787Crossref PubMed Scopus (69) Google Scholar, M. Myslinski E. Schuster C. Krol A. Carbon P. EMBO J. 1997; 16: 173-181Crossref PubMed Scopus (90) Google Scholar, C. Krol A. Carbon P. Mol. Cell. Biol. 1998; 18: 2650-2658Crossref PubMed Scopus (33) Google Scholar, E. Krol A. Carbon P. J. Biol. Chem. 1998; 273: 21998-22006Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). of the activation of Staf, its activation domains, have (6Schuster C. Krol A. Carbon P. Mol. Cell. Biol. 1998; 18: 2650-2658Crossref PubMed Scopus (33) Google Scholar, E. Krol A. Carbon P. J. Biol. Chem. 1998; 273: 21998-22006Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), is known the molecular basis of the interaction with its sites on the This is the first one a analysis of the of interaction between the zinc DNA binding domain of Staf and different Staf-responsive In we in this all of the seven zinc fingers are required to establish DNA The is that zinc finger 7 does not establish base-specific contacts with the The with zinc finger 1 is it does establish contacts with the DNA in the Xenopus tRNASec, but not in the human U6 promoter. have a of binding site selection from a of to the high affinity consensus binding site ATTACCCATAATGCATYGCGG for Staf. This consensus shows to known for other zinc finger proteins of the C2-H2 type. The selection data demonstrated a for an at with an at and a C residue at In the central part of the the in our It is to that the seven highly and all reside at the in the majority of the Staf-responsive elements (5Schaub M. Myslinski E. Schuster C. Krol A. Carbon P. EMBO J. 1997; 16: 173-181Crossref PubMed Scopus (90) Google Scholar). the C at and are conserved in all the identified Staf-responsive In the of it is to that or the on the establish that are for the binding affinity and The that can is the consensus binding site constitutes the Staf-responsive In to the affinities of the zinc finger domain found that the optimal sequence a affinity the Staf-responsive elements in the Xenopus human U6 snRNA promoters. However, the affinity of the protein for Xenopus tRNASec and human U6 sites that of its truncated containing only the DNA binding domain (7Myslinski E. Krol A. Carbon P. J. Biol. Chem. 1998; 273: 21998-22006Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The for the affinity of the zinc finger domain to be it is a well known for of the that the protein of the zinc finger domain the differences in DNA binding affinities J. Mol. Cell. Biol. 1995; PubMed Scopus Google Scholar). binding of Staf to the DNA other of the not to the zinc finger domain but to a to the of found that Staf is to bind specifically with a relative high affinity to a of divergent DNA in the sequence elements of and gene promoters and M. Myslinski E. Schuster C. Krol A. Carbon P. EMBO J. 1997; 16: 173-181Crossref PubMed Scopus (90) Google Scholar). the amino sequence is conserved the seven zinc finger domain between the Xenopus and human Staf (7Myslinski E. Krol A. Carbon P. J. Biol. Chem. 1998; 273: 21998-22006Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), the specific DNA by Staf in the Xenopus tRNASec and human U6 snRNA promoters are clearly only The selection that 1, and 21 This the sequence in the Staf-responsive where only two of these seven are conserved between the Xenopus tRNASec and human U6 sites. a one can how DNA binding domains can contact such divergent regulatory DNA sequences. The from the work, in which we have that Staf zinc finger 1 in a flexible in to to the divergent elements in the Xenopus tRNASec and human U6 promoters. Two sets of in this First, of the for the containing that of zinc finger 1 a effect on the affinity for the Xenopus tRNASec for the human U6 sites. of the from the and binding site selection revealed that zinc finger 1 is involved in the DNA in the Xenopus tRNASec but not in the human U6 promoter. the presence of zinc finger 1 the of the on the Xenopus tRNASec that of an of 7 and on the and respectively. It is that the between the of and a of not only by zinc finger 1 but also by the zinc finger that the of can be for by the lack or interaction zinc finger and the DNA, in the of zinc finger The that Staf a of contacts the DNA In the lack of in the from one to the other is with the protein a to that of the In the of the complexes with and the C2-H2 fingers of these proteins bind in the of the DNA, contacts with one of the (9Pavletich N.P. Pabo C.O. Science. 1991; 252: 809-817Crossref PubMed Scopus (1763) Google Scholar, 10Fairall R. Schwabe J.W.R. Chapman L. Finch J.T. Rhodes D. Nature. 1993; 366: 483-487Crossref PubMed Scopus (300) Google Scholar, 11Pavletich N.P. Pabo C.O. Science. 1993; 261: 1701-1707Crossref PubMed Scopus (613) Google Scholar, 12Elrod-Erickson M. Rould M.A. Nekludova L. Pabo C.O. Structure. 1996; 4: 1171-1180Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar). it is that in the Staf-responsive elements of the human U6 and Xenopus tRNASec promoters, Staf the of the DNA the on the on the Staf with the of the Staf-responsive that zinc finger 7 is not involved in the in the Staf-responsive however, to Staf-responsive elements This can be to that zinc finger 7 is involved in contacts with the of the Staf binding sites. zinc finger 7 can be involved in an conserved that of In this the of the has established that zinc finger 1 of does not contact the DNA but has with the zinc finger N.P. Pabo C.O. Science. 1993; 261: 1701-1707Crossref PubMed Scopus (613) Google Scholar). the binding to the Xenopus utilizes six of the seven zinc fingers, whereas are for Staf to bind to the human U6 site. It is that it sequence zinc finger 1 is not with all promoters. it is to this flexible that Staf can bind to divergent the promoters of snRNA-type in different this to recognize different binding site Staf to gene from a of different promoters. are to E. Myslinski and C. Schuster for of the and to C. for