Sashidhar Mulugu

In budding yeast, phosphate starvation triggers inhibition of the Pho80-Pho85 cyclin-cyclin-dependent kinase (CDK) complex by the CDK inhibitor Pho81, leading to expression of genes involved in nutrient homeostasis. We isolated myo-d-inositol heptakisphosphate (IP7) as a cellular component that stimulates Pho81-dependent inhibition of Pho80-Pho85. IP7 is necessary for Pho81-dependent inhibition of Pho80-Pho85 in vitro. Moreover, intracellular concentrations of IP7 increased upon phosphate starvation, and yeast mutants defective in IP7 production failed to inhibit Pho80-Pho85 in response to phosphate starvation. These observations reveal regulation of a cyclin-CDK complex by a metabolite and suggest that a complex metabolic network mediates signaling of phosphate availability.

Inositol pyrophosphates are a diverse group of high-energy signaling molecules whose cellular roles remain an active area of study. We report a previously uncharacterized class of inositol pyrophosphate synthase and find it is identical to yeast Vip1 and Asp1 proteins, regulators of actin-related protein-2/3 (ARP 2/3) complexes. Vip1 and Asp1 acted as enzymes that encode inositol hexakisphosphate (IP6) and inositol heptakisphosphate (IP7) kinase activities. Alterations in kinase activity led to defects in cell growth, morphology, and interactions with ARP complex members. The functionality of Asp1 and Vip1 may provide cells with increased signaling capacity through metabolism of IP6.

Using yeast forward and reverse two-hybrid analyses, we have discovered that the replication terminator protein Tus of Escherichia coli physically interacts with DnaB helicase in vivo. We have confirmed this protein-protein interaction in vitro. We show further that replication termination involves protein-protein interaction between Tus and DnaB at a critical region of Tus protein, called the L1 loop. Several mutations located in the L1 loop region not only reduced the protein-protein interaction but also eliminated or reduced the ability of the mutant forms of Tus to arrest DnaB at a Ter site. At least one mutation, E49K, significantly reduced Tus-DnaB interaction and almost completely eliminated the contrahelicase activity of Tus protein in vitro without significantly reducing the affinity of the mutant form of Tus for Ter DNA, in comparison with the wild-type protein. The results, considered along with the crystal structure of Tus-Ter complex, not only elucidate further the mechanism of helicase arrest but also explain the molecular basis of polarity of replication fork arrest at Ter sites.

The current models that have been proposed to explain the mechanism of replication termination are (i) passive arrest of a replication fork by the terminus (Ter) DNA-terminator protein complex that impedes the replication fork and the replicative helicase in a polar fashion and (ii) an active barrier model in which the Ter-terminator protein complex arrests a fork not only by DNA-protein interaction but also by mechanistically significant terminator protein-helicase interaction. Despite the existence of some evidence supporting in vitro interaction between the replication terminator protein (RTP) and DnaB helicase, there has been continuing debate in the literature questioning the validity of the protein-protein interaction model. The objective of the present work was two-fold: (i) to reexamine the question of RTP-DnaB interaction by additional techniques and different mutant forms of RTP, and (ii) to investigate if a common domain of RTP is involved in the arrest of both helicase and RNA polymerase. The results validate and confirm the RTP-DnaB interaction in vitro and suggest a critical role for this interaction in replication fork arrest. The results also show that the Tyr33 residue of RTP plays a critical role both in the arrest of helicase and RNA polymerase. The current models that have been proposed to explain the mechanism of replication termination are (i) passive arrest of a replication fork by the terminus (Ter) DNA-terminator protein complex that impedes the replication fork and the replicative helicase in a polar fashion and (ii) an active barrier model in which the Ter-terminator protein complex arrests a fork not only by DNA-protein interaction but also by mechanistically significant terminator protein-helicase interaction. Despite the existence of some evidence supporting in vitro interaction between the replication terminator protein (RTP) and DnaB helicase, there has been continuing debate in the literature questioning the validity of the protein-protein interaction model. The objective of the present work was two-fold: (i) to reexamine the question of RTP-DnaB interaction by additional techniques and different mutant forms of RTP, and (ii) to investigate if a common domain of RTP is involved in the arrest of both helicase and RNA polymerase. The results validate and confirm the RTP-DnaB interaction in vitro and suggest a critical role for this interaction in replication fork arrest. The results also show that the Tyr33 residue of RTP plays a critical role both in the arrest of helicase and RNA polymerase. replication terminator protein 4-morpholinepropanesulfonic acid S-[N-(4-azidosalicyl)cysteaminyl]-2-thiopyridine glutathione S-transferase DNA replication in many prokaryotic chromosomes and at some eukaryotic chromosome regions is arrested at specific sequences called replication termini or Ter sites (6Bussiere D.E. Bastia D. Mol. Microbiol. 1999; 31: 1611-1618Crossref PubMed Scopus (73) Google Scholar). Ter sites act as polar barriers to fork movement and act essentially as replication traps. In Bacillus subtilis, the TerDNA interacts with a sequence-specific DNA-binding protein called replication terminator protein (RTP)1 that acts as a polar contrahelicase; i.e. it impedes helicase-catalyzed DNA unwinding when present in one orientation, whereas it lets the helicase pass through unimpeded in the opposite orientation (1Khatri G.S. MacAllister T. Sista P. Bastia D. Cell. 1989; 59: 667-674Abstract Full Text PDF PubMed Scopus (143) Google Scholar, 2Lee E.H. Kornberg A. Hidaka M. Kobayashi T. Horiuchi T. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9104-9108Crossref PubMed Scopus (129) Google Scholar, 3Hill T.M. Marians K.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2481-2485Crossref PubMed Scopus (107) Google Scholar, 4Kaul S. Mohanty B.K. Sahoo T. Patel I. Khan S. Bastia D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11143-11147Crossref PubMed Scopus (37) Google Scholar). The bacterial chromosome is believed to exist in vivo not as naked DNA but as a DNA-protein complex, with most of the DNA-binding proteins remaining bound to the chromosome (5Yamamoto K.R. Alberts B.M. Annu. Rev. Biochem. 1976; 45: 721-746Crossref PubMed Scopus (422) Google Scholar). Despite the fact that some of these proteins bind to DNA with relatively high affinity (e.g. lac repressor), the replication fork apparently has the ability to pass through these complexes unimpeded. The only region of the chromosome that is known to arrest effectively the replication forks is the terminus (6Bussiere D.E. Bastia D. Mol. Microbiol. 1999; 31: 1611-1618Crossref PubMed Scopus (73) Google Scholar). The preceding observations suggest the following: (i) the replication apparatus apparently has an activity that allows it to pass through most protein-DNA complexes, some of which contain strong DNA-binding proteins, and (ii) since the replication terminus is able to arrest forks effectively, the terminator protein-DNA complex is likely to have special features that enable it to arrest replication forks. Thus, high affinity binding of terminator protein to Ter sites per se does not appear to be sufficient to cause the replication-terminating activity of RTP (12Gautam A. Bastia D. J. Biol. Chem. 2001; 276: 8771-8777Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). We have hypothesized that DnaB (or the equivalent helicase of B. subtilis)-RTP interaction plays a key role in fork arrest (1Khatri G.S. MacAllister T. Sista P. Bastia D. Cell. 1989; 59: 667-674Abstract Full Text PDF PubMed Scopus (143) Google Scholar). Despite the existence of in vitro evidence for RTP-DnaB interaction (7Manna A.C. Pai K.S. Bussiere D.E. Bastia D. Cell. 1996; 87: 881-891Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 12Gautam A. Bastia D. J. Biol. Chem. 2001; 276: 8771-8777Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar), the validity of the protein-protein interaction model has been debated (11Duggin I.G. Anderson P.A. Smith M.T. Wilce J.A. King G.F. Wake R.G. J. Mol. Biol. 1999; 266: 1325-1335Crossref Scopus (23) Google Scholar). The raison d'etre for carrying out this work was 2-fold: (i) to perform additional experiments, using independent approaches and different mutant forms of RTP, to reexamine the question of RTP-DnaB interaction in vitro and (ii) to investigate whether a common domain of RTP is involved in the arrest of both RNA polymerase and helicase. The observations presented here confirm the biologically meaningful interaction between RTP and DnaB and further extend the result by showing that a common domain of RTP seems to be involved in the arrest of both DnaB helicase and T7 RNA polymerase (and perhaps other RNA polymerases). The replication termini of B. subtilis (Fig.1) consist of overlapping core and auxiliary sites. A RTP dimer first binds to a core and then, by cooperativity, promotes the binding of a second dimer of RTP to the auxiliary site (8Pai K.S. Bussiere D.E. Wang F. Hutchison C. White S.W. Bastia D. EMBO J. 1996; 15: 3164-3317Crossref PubMed Scopus (22) Google Scholar, 9Bussiere D.E. Bastia D. White S.W. Cell. 1995; 80: 651-660Abstract Full Text PDF PubMed Scopus (78) Google Scholar). Interaction between two dimers is essential for fork arrest with the core end of the Ter site arresting the helicase and the auxiliary end, allowing the helicase to pass through unimpeded (7Manna A.C. Pai K.S. Bussiere D.E. Bastia D. Cell. 1996; 87: 881-891Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The RTP of B. subtilis is a homodimer belonging to the class of winged helix proteins (8Pai K.S. Bussiere D.E. Wang F. Hutchison C. White S.W. Bastia D. EMBO J. 1996; 15: 3164-3317Crossref PubMed Scopus (22) Google Scholar, 9Bussiere D.E. Bastia D. White S.W. Cell. 1995; 80: 651-660Abstract Full Text PDF PubMed Scopus (78) Google Scholar). The crystal structure of the RTP apoprotein has been solved at high resolution, and the structure contains four α-helices, three β strands, and an unstructured, N-terminal arm (Fig. 2 A) (9Bussiere D.E. Bastia D. White S.W. Cell. 1995; 80: 651-660Abstract Full Text PDF PubMed Scopus (78) Google Scholar). Extensive random and site-directed mutagenesis of RTP had identified the N-terminal arm, the α3 helix, and the β2 strand to be the main DNA-binding elements (8Pai K.S. Bussiere D.E. Wang F. Hutchison C. White S.W. Bastia D. EMBO J. 1996; 15: 3164-3317Crossref PubMed Scopus (22) Google Scholar), with the α3 inserting into the major groove and the β2 into the minor groove of Ter DNA (8Pai K.S. Bussiere D.E. Wang F. Hutchison C. White S.W. Bastia D. EMBO J. 1996; 15: 3164-3317Crossref PubMed Scopus (22) Google Scholar). Affinity cleavage analysis that converted RTP to a site-directed chemical nuclease was used to determine amino acid to base contacts, and the results had confirmed that the α3 helix contacted the major groove and β2, to the minor groove of Ter DNA (10Pai K.S. Bussiere D.E. Wang W. White S.W. Bastia D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10647-10652Crossref PubMed Scopus (19) Google Scholar, 26Mohanty B.K. Bussiere D.E. Sahoo T. Pai K.S. Meijer W, J.J. Bron S. Bastia D. J. Biol. Chem. 2001; 276: 13160-13168Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). X-ray crystallography had revealed an exposed hydrophobic patch that was suggested to be a possible docking surface for the helicase (9Bussiere D.E. Bastia D. White S.W. Cell. 1995; 80: 651-660Abstract Full Text PDF PubMed Scopus (78) Google Scholar) (see Fig.2 B). In this paper, we have used cross-linking, label transfer, and other techniques along with different mutant forms of RTP to present additional evidence in favor of the RTP-DnaB interaction model of replication termination. We also show that a common region of RTP is involved in arresting both the helicase and T7 RNA polymerase. Both helicase and RNA polymerases melt DNA, and the results suggest that there probably is a common structural motif in these enzymes that may be recognized by RTP. The Escherichia coli strains JM109 (sup E44, rel A1,recA, endA1, gyrA96,hsdR17D, Δ[lac, proAB], [F‘tra D36, lacIqΔ(lacZ)M15,proA+,rk−,mk−B+) was used for making M13 DNA; DH5α [F‘sup E44,lacU169 (φ80 lacZ Δ M15), hsdR17,recA1, endA1, gyrA96,thi-1, relA1] was used for cloning; and BL21{DE3}[F− omp T, hsd S, rB−,mB- gal] containing the plasmid pLysS was used for expressing proteins in pET vectors (Novagen). Wild was used for of in in vitro replication The out as in A.C. Pai K.S. Bussiere D.E. White Bastia D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: PubMed Scopus Google polymerase and core binding sites RTP binding site and the core binding site of RTP using M13 and with and of DNA was used in which was out in of of DNA, and of RTP and The out at for and to and DNA was by a these the of bound by two dimers of RTP protein was and as a of the RTP (see The as the (Fig. a of the T. Mohanty B.K. Bastia D. EMBO J. 1995; PubMed Scopus Google Scholar). is the is a to the of RTP at in this the for between is the the is 2 allowing of the of RTP for are the and the 2 for of by and replication terminator are the for and the of RTP for of DNA analysis of RTP, RTP and RTP in a The proteins as (7Manna A.C. Pai K.S. Bussiere D.E. Bastia D. Cell. 1996; 87: 881-891Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, K.S. Bussiere D.E. Wang W. White S.W. Bastia D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10647-10652Crossref PubMed Scopus (19) Google Scholar). as in G.S. MacAllister T. Sista P. Bastia D. Cell. 1989; 59: 667-674Abstract Full Text PDF PubMed Scopus (143) Google of with RTP binding sites using and and to of binding site in orientation or M13 binding site in orientation DNA, helicase through a of used in of containing 2 and of RTP and of at of DnaB helicase was to the and was for at The was with and In vitro replication out to 3Hill T.M. Marians K.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2481-2485Crossref PubMed Scopus (107) Google with some coli of binding site in and binding site in M. 1995; PubMed Scopus Google Scholar) plasmid used in of of and of of and a of RTP was used in the RTP was to bind to site at for and was to and the at for The was by to and to was out at for by for and of replication The replication and an was used as a The was to mutant and RTP proteins to Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). and mutant forms of RTP by using the site-directed mutagenesis and proteins to the 1994; PubMed Scopus Google Scholar). of protein was used for in a containing and The was at for by at was by the and for at of proteins with DNA Ter RTP binding site out in a containing a or of proteins RTP, and of DNA, and of The was in the at for to was out using the of the at a of for 2 The affinity to A.C. Pai K.S. Bussiere D.E. Bastia D. Cell. 1996; 87: 881-891Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 1994; PubMed Scopus Google Scholar, and Mohanty B.K. M. Bastia D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: Scopus Google in the with an in of and S-[N-(4-azidosalicyl)cysteaminyl]-2-thiopyridine in was and in in the and at was out in a containing of in and of The was to an and by the to a containing of and in a at the was used to with RTP of the with of RTP in and for The was 2 of in and in the containing and used for of of DnaB helicase, and of and using a model for at in the of for and for at in the The was at to using of in to and for at in the of the of and was and to the second and was and the In vitro out as M. 1995; PubMed Scopus Google Scholar, D. Mohanty B.K. M. DNA in Scholar, Biochem. J. PubMed Google Scholar, B.K. Sahoo T. Bastia D. EMBO J. 1996; 15: PubMed Scopus Google Scholar). Despite the existence of some evidence for RTP-DnaB vitro (7Manna A.C. Pai K.S. Bussiere D.E. Bastia D. Cell. 1996; 87: 881-891Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) and in vivo (12Gautam A. Bastia D. J. Biol. Chem. 2001; 276: 8771-8777Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar), there has been continuing debate (11Duggin I.G. Anderson P.A. Smith M.T. Wilce J.A. King G.F. Wake R.G. J. Mol. Biol. 1999; 266: 1325-1335Crossref Scopus (23) Google Scholar) as to whether RTP-DnaB helicase is involved in replication fork arrest. debate to reexamine this significant question by using different approaches and additional a mutant of RTP. Tyr33 was for further work had this as a critical residue for helicase arrest (7Manna A.C. Pai K.S. Bussiere D.E. Bastia D. Cell. 1996; 87: 881-891Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). We also to investigate whether the domain of RTP that is known to be involved in helicase arrest also impedes the of RNA polymerase. we to determine if the Tyr33 residue of RTP was in with Ter DNA in as The exposed hydrophobic patch of RTP was to be a possible docking surface for DnaB helicase (9Bussiere D.E. Bastia D. White S.W. Cell. 1995; 80: 651-660Abstract Full Text PDF PubMed Scopus (78) Google Scholar), and the Tyr33 the was to be a critical residue in the RTP-DnaB interaction in R.G. King Full Text Full Text PDF PubMed Scopus (19) Google Scholar). the crystal structure of an complex is not we to investigate whether the Tyr33 residue contacted Ter DNA in We the by site-directed mutagenesis and the to RTP has a residue in the at and mutant forms of RTP. was for (Fig. A) the residue is in the α3 helix of RTP (Fig. 2 A) and is known to the major groove DNA (10Pai K.S. Bussiere D.E. Wang W. White S.W. Bastia D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10647-10652Crossref PubMed Scopus (19) Google Scholar, 26Mohanty B.K. Bussiere D.E. Sahoo T. Pai K.S. Meijer W, J.J. Bron S. Bastia D. J. Biol. Chem. 2001; 276: 13160-13168Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). was used as a RTP the significant of with DNA (Fig. and In protein of protein-DNA and two to DNA bound to a dimer or with two dimers of RTP, (Fig. Thus, the of the result presented we that the contacted DNA in was of the Ter DNA of the is P. P. EMBO J. PubMed Scopus Google Scholar). the of the interaction between RTP and DnaB we DnaB with a at the N-terminal end and the protein to and it with and T. Mohanty B.K. Bastia D. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). work had used DnaB by in vitro (7Manna A.C. Pai K.S. Bussiere D.E. Bastia D. Cell. 1996; 87: 881-891Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). We used DnaB in vivo that had helicase activity not in to of binding by or DnaB Wild RTP and the mutant forms as proteins with and We that of and of the mutant forms of RTP to a of the affinity by of of RTP affinity and the of bound proteins by We the the protein and to that the protein was biologically be in that the Tyr33 residue out of the surface of RTP apoprotein (Fig. 2 and at this site does not to the of the protein as suggested by that that of the In we able to the mutant forms and a of but not to the of DnaB protein bound to equivalent of the and mutant forms of the and the protein was with of in and The of bound proteins was with a The results that the RTP the the fact that RTP is a whereas DnaB is the of and protein interaction was into and by the We and protein and the affinity with RTP affinity We not of by affinity interaction binding is also not by that of an Tyr33 residue by or A of the but the protein-protein interaction. In of or in a in protein-protein interaction (Fig. A and B). The of the affinity binding with the that there was specific protein-protein interaction between RTP and DnaB in and in the of Ter DNA and that the Tyr33 residue a key role in that interaction. We also to reexamine possible interaction of DnaB to RTP by an independent We the T. Mohanty B.K. Bastia D. EMBO J. 1995; PubMed Scopus Google Scholar) at to the and it to RTP at the residue 2 was the crystal structure that both and to other the protein surface and that both out at the protein at residue only RTP-DnaB interaction (7Manna A.C. Pai K.S. Bussiere D.E. Bastia D. Cell. 1996; 87: 881-891Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). RTP a at in the and the mutant of RTP also in the with The of the is in A. RTP with the protein to cleavage of the by with the label to The RTP label to DnaB the at is in the domain in the helix the Tyr33 residue a critical with DnaB as suggested by the affinity the mutant with at the label to The results of the in the of Ter DNA, are in and C. The RTP, with the of the the in the domain was We with DnaB and the with to the The RTP, to a complex with a in a (Fig. of the with cleavage at the and label the RTP to DnaB as by a of that with DnaB in the (Fig. the was not with label to DnaB (Fig. be that the in between the complex is and it a in an to the A of the but the RTP-DnaB complex to at a that was that of DnaB (Fig. 2 and We to determine whether the interaction between the two proteins was specific and if the Tyr33 residue a critical role in the interaction. The results that when the mutant of RTP was by the label in a complex or in DnaB (Fig. 2 The mutant to show label (Fig. 2 and Thus, the cross-linking, label with the the affinity binding that RTP in vitro with DnaB in the of Ter The in vitro between RTP and DnaB was also out in the of Ter DNA, and the results to that in the of DNA that the of label was We to determine further the of DnaB that was the of the label but to to the of determine the DNA binding and of binding of and the and mutant forms of RTP, The results these are in as the of DNA as a of the of the RTP three forms of the RTP bound to DNA with high with showing some in affinity The of RTP for two dimers of of and for the and RTP binding by the and RTP forms was as be the in bound with RTP and as in the (Fig. The for binding by and and In binding by the RTP of high was Thus, the results that the not or only the and binding affinity of the mutant of the protein DNA, it RTP-DnaB We have the of protein in with that of RTP and have that the mutant had a that of the protein not to the ability of the and the Tyr33 mutant of RTP to DnaB helicase two M13 containing and of Ter to to that the Ter site at the The ability of DnaB helicase to melt and the the in the of of and the mutant of RTP was Wild RTP, as was able to arrest DnaB in a polar whereas the mutant was in this result that is involved in interaction with DnaB as a also in helicase arrest in In vitro replication with coli out with DNA binding site in both with to Wild RTP and the RTP mutant forms for ability to arrest the replication fork The replication The of a to strand of replication was of fork arrest by RTP. The orientation of Ter the arrested replication in the of RTP, but the was not in the orientation of Ter RTP The and RTP to arrest replication showing an of this (Fig. and Thus, the to at the residue of RTP in a of replication fork arresting work had that RTP arrested RNA polymerases and coli RNA in a polar We to investigate whether a common region of RTP was involved in the arrest of both and RNA We using a that had an T7 and a Ter site in orientation with to the the was arrested in the orientation of the by RTP. mutant forms of RTP, and to arrest T7 RNA polymerase in the orientation of the Ter site the Ter site in the opposite orientation as to arrest (Fig. Thus, it that the Tyr33 residue is a key not only in arresting helicase-catalyzed DNA unwinding but also in RNA the ability of replication terminator proteins to arrest replicative and RNA polymerase has been known for some (1Khatri G.S. MacAllister T. Sista P. Bastia D. Cell. 1989; 59: 667-674Abstract Full Text PDF PubMed Scopus (143) Google Scholar, 2Lee E.H. Kornberg A. Hidaka M. Kobayashi T. Horiuchi T. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9104-9108Crossref PubMed Scopus (129) Google Scholar, 4Kaul S. Mohanty B.K. Sahoo T. Patel I. Khan S. Bastia D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11143-11147Crossref PubMed Scopus (37) Google Scholar) and we have in vitro evidence for RTP-DnaB interaction (6Bussiere D.E. Bastia D. Mol. Microbiol. 1999; 31: 1611-1618Crossref PubMed Scopus (73) Google Scholar), the of the mechanism by which RTP arrests replication fork and additional The of the RTP-DnaB interaction by independent and different was also in by continuing debate in the literature as to whether RTP-DnaB interaction was involved in replication fork arrest (11Duggin I.G. Anderson P.A. Smith M.T. Wilce J.A. King G.F. Wake R.G. J. Mol. Biol. 1999; 266: 1325-1335Crossref Scopus (23) Google Scholar). had that the in the had a in the DNA binding affinity and that this in DNA-protein interaction the in RTP-DnaB interaction was for to arrest replication forks. present results show that the strong and binding of RTP to DNA as by the The show a in affinity for a of this to of interaction the activity of The to be in the of RTP, one in the Ter site to affinity for RTP and whether a is of arresting forks in We have this of by the mechanism of fork arrest at the Ter site of B. The site binds to DNA with a of the DNA-protein complex of a whereas the Ter complex has a of Despite this in the of the two DNA protein complexes, we that the was able to arrest replication forks in that strong was not for fork arrest (12Gautam A. Bastia D. J. Biol. Chem. 2001; 276: 8771-8777Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). be that of the evidence for RTP-DnaB interaction presented here and is of an in (7Manna A.C. Pai K.S. Bussiere D.E. Bastia D. Cell. 1996; 87: 881-891Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). in vivo we have to perform analysis using RTP and DnaB but have been to of of RTP in we have been in showing the in vivo interaction between terminator protein and DnaB of coli using the using a we have mutant forms of that bind to Ter but are in interaction with DnaB and in helicase arrest. A. S. J. and D. the protein and RTP have different crystal the of both proteins are that by S. Mohanty B.K. Sahoo T. Patel I. Khan S. Bastia D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11143-11147Crossref PubMed Scopus (37) Google Scholar, D.E. Bastia D. Mol. Microbiol. 1999; 31: 1611-1618Crossref PubMed Scopus (73) Google Scholar, 9Bussiere D.E. Bastia D. White S.W. Cell. 1995; 80: 651-660Abstract Full Text PDF PubMed Scopus (78) Google Scholar). The results subtilis and coli a mechanism of replication termination that not only Ter-terminator protein interaction but also mechanistically significant terminator protein-helicase interaction. analysis of the helicase an model of helicase that DNA and helicase DNA P. P. EMBO J. PubMed Scopus Google Scholar). work be able to whether terminator proteins both of these by the helicase. the of mutant forms of RTP with amino acid at Tyr33 to arrest helicase and RNA polymerase suggest a common surface RTP. be in the to mutant forms of T7 RNA polymerase that arrest by RTP. mutant forms of T7 RNA along with the crystal structure of the D. EMBO J. PubMed Scopus Google Scholar), be a for further and the mechanism that arrest of RNA We of for a of the