Kaiyu Ma

We have examined the behavior of T7 RNA polymerase (RNAP) at a set of promoter variants having all possible single base pair (bp) substitutions. The polymerase exhibits an absolute requirement for initiation with a purine and a strong preference for initiation with GTP vs ATP. Promoter variants that would require initiation at the normal start site (+1) with CTP or UTP result in a shift in initiation to +2 (with GTP). However, the choice of start site is little affected by base substitutions elsewhere in the initiation region. Furthermore, when the initiation region is shifted either one nucleotide (nt) closer or 1 nt further away from the binding region, transcription still begins the same distance downstream. These results indicate that the sequence around the start site is of little importance in start site selection and that initiation is directed a minimum distance of 5 nt downstream from the binding region. At promoters that initiate with +1 GGG, T7 RNAP synthesizes a ladder of poly(G) products as a result of slippage of the transcript on the three C residues in the template strand from +1 to +3. At promoter variants in which there is an opportunity to form a longer RNA-DNA hybrid, this G-ladder is enhanced and extended. This observation is not in agreement with recent suggestions that the RNA-DNA hybrid in the initiation complex cannot extend further than 3 bps upstream from the active site [Cheetham, G., Jeruzalmi, D., and Steitz, T. A. (1999) Nature 399, 80-83].

Heel pain is a very common foot disease. Varieties of names such as plantar fasciitis, jogger's heel, tennis heal, policeman's heel are used to describe it. Mechanical factors are the most common etiology of heel pain. Common causes of hell pain includes: Plantar Fasciitis, Heel Spur, Sever's Disease, Heel bump, Achilles Tendinopathy, Heel neuritis, Heel bursitis. The diagnosis is mostly based on clinical examination. Normally, the location of the pain and the absence of associated symptoms indicating a systemic disease strongly suggest the diagnosis. Several therapies exist including rest, physical therapy, stretching, and change in footwear, arch supports, orthotics, night splints, anti-inflammatory agents, and surgery. Almost all patients respond to conservative nonsurgical therapy. Surgery is the last treatment option if all other treatments had failed. Rest, ice, massage, the use of correct exercise and complying with a doctor's advice all play important part in helping to recover from this hell pain condition, but getting good quality, suitable shoes with the appropriate amount of support for the whole foot is the most important.

During the early stages of transcription, T7 RNA polymerase forms an unstable initiation complex that synthesizes and releases transcripts 2-8 nt in length before disengaging from the promoter and isomerizing to a stable elongation complex. In this study, we used RNA small middle dotprotein and RNA small middle dotDNA crosslinking methods to probe the location of newly synthesized RNA in halted elongation complexes. The results indicate that the RNA in an elongation complex remains in an RNA small middle dotDNA hybrid for about 8 nt from the site of nucleotide addition and emerges to the surface of the enzyme about 12 nt from the addition site. Strikingly, as the transcript leaves its hybrid with the template, the crosslinks it forms with the RNA polymerase involve a portion of a hairpin loop (the specificity loop) that makes specific contacts with the binding region of the promoter during initiation. This observation suggests that the specificity loop may have a dual role in transcription, binding first to the promoter and subsequently interacting with the RNA product. It seems likely that association of the nascent RNA with the specificity loop facilitates disengagement from the promoter and is an important part of the process that leads to a stable elongation complex.

To examine changes that occur during the transition from an initiation complex (IC) to an elongation complex (EC) in T7 RNA polymerase (RNAP), we used nucleic acid-protein cross-linking methods to probe interactions of the RNAP with RNA and DNA in a halted EC. As the RNA is displaced from the RNA-DNA hybrid ∼9 bp upstream from the active site (at −9) it interacts with a region within the specificity loop (residues 744–750) and is directed toward a positively charged surface that surrounds residues Lys-302 and Lys-303. Surprisingly, the template and non-template strands of the DNA at the upstream edge of the hybrid (near the site where the RNA is displaced) interact with a region in the N-terminal domain of the RNAP (residues 172–191) that is far away from the specificity loop before isomerization (in the IC). To bring these two regions of the RNAP into proximity, major conformational changes must occur during the transition from an IC to an EC. The observed nucleic acid-protein interactions help to explain the behavior of a number of mutant RNAPs that are affected at various stages in the initiation process and in termination. To examine changes that occur during the transition from an initiation complex (IC) to an elongation complex (EC) in T7 RNA polymerase (RNAP), we used nucleic acid-protein cross-linking methods to probe interactions of the RNAP with RNA and DNA in a halted EC. As the RNA is displaced from the RNA-DNA hybrid ∼9 bp upstream from the active site (at −9) it interacts with a region within the specificity loop (residues 744–750) and is directed toward a positively charged surface that surrounds residues Lys-302 and Lys-303. Surprisingly, the template and non-template strands of the DNA at the upstream edge of the hybrid (near the site where the RNA is displaced) interact with a region in the N-terminal domain of the RNAP (residues 172–191) that is far away from the specificity loop before isomerization (in the IC). To bring these two regions of the RNAP into proximity, major conformational changes must occur during the transition from an IC to an EC. The observed nucleic acid-protein interactions help to explain the behavior of a number of mutant RNAPs that are affected at various stages in the initiation process and in termination. RNA polymerase DNA polymerase initiation complex elongation complex nucleotides wild type hydroxylamine 2-nitro-5-thiocyano-benzoic acid N-chlorosuccinimide 4-thio-UMP template non-template 4-morpholinepropanesulfonic acid 4-morpholineethanesulfonic acid iron (S)-1-(p-bromoacetamidobenzyl)ethylenediamine-tetraacetate endoproteinase GluC (protease V8) E. coli outer membrane protease OmpT 4-thio-dTMP Like all RNA polymerases (RNAPs),1 T7 RNAP forms an unstable initiation complex (IC) that synthesizes and releases short abortive products before clearing the promoter and forming a stable elongation complex (EC) (Ref. 1Martin C.T. Muller D.K. Coleman J.E. Biochemistry. 1988; 27: 3966-3974Crossref PubMed Scopus (235) Google Scholar; for review, see Ref. 2McAllister W.T. Nucleic Acids Mol. Biol. 1997; 11: 15-25Crossref Google Scholar). The transition is accompanied by release of promoter contacts, changes in the size of the footprint of the polymerase on the DNA, and changes in accessibility to cleavage by a variety of proteases (3Ikeda R.A. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 3614-3618Crossref PubMed Scopus (147) Google Scholar, 4Place C. Oddos J. Buc H. W.T. Biochemistry. Scopus Google Scholar, J. PubMed Scopus Google Scholar, J. Mol. Biol. PubMed Scopus Google Scholar). these changes that in the of the complex in the of the occur during the and the behavior of mutant and the of changes in template on abortive that during the that from to and a from ∼9 to J. PubMed Scopus Google J. Mol. Biol. 1997; PubMed Scopus Google Scholar, Biochemistry. 1997; PubMed Scopus Google Scholar, W.T. J. Mol. Biol. PubMed Scopus Google Scholar, W.T. J. Mol. Biol. PubMed Scopus Google Scholar, C.T. W.T. J. Mol. Biol. Scopus Google Scholar, C. C.T. J. Biol. PubMed Scopus Google Scholar). the stages to the upstream region of the promoter and the RNAP are the active site the RNA-DNA hybrid a of bp J. PubMed Scopus Google Scholar, C. C.T. J. Biol. PubMed Scopus Google J. J. Mol. Biol. PubMed Scopus Google the of the hybrid to to bp before the to a hybrid of is observed in the C. C.T. J. Biol. PubMed Scopus Google Scholar). cross-linking that stages during promoter at and are before the at C. Oddos J. Buc H. W.T. Biochemistry. Scopus Google Scholar). the the RNA-DNA hybrid is bp and is in a of ∼9 bp J. J. Mol. Biol. PubMed Scopus Google Scholar, A. S. W.T. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, C. C.T. J. Mol. Biol. PubMed Scopus Google Scholar). The upstream of the is to the at the RNA is displaced from the and the is to the of the RNA C. C.T. J. Mol. Biol. PubMed Scopus Google Scholar). As the RNA is displaced from the hybrid at it with a region of the RNAP specificity that is in promoter at the of the RNA from of interactions with the RNAP J. J. Mol. Biol. PubMed Scopus Google Scholar, A. S. W.T. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google for for RNAP with a of for a and for an IC that the in the template PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). for an for that during the transition from an IC to an EC. we the of the T7 RNAP the of nucleic acid-protein cross-linking The are with of the and that major the N-terminal domain occur during the transition to an a number of of T7 RNAP the of a T7 RNAP EC. The in in the of the by nucleic cross-linking are with the of a T7 RNAP IC and that major conformational changes must occur during the transition from an IC to an and PubMed Scopus Google the of a T7 RNAP IC in the of the template the upstream promoter that observed in the T7 complex (in the of are the active site the in of the of the into a that the with to the release of abortive products and promoter during the of J. PubMed Scopus Google C.T. W.T. J. Mol. Biol. Scopus Google Scholar, PubMed Scopus Google the of the and PubMed Scopus Google that of the RNA-DNA hybrid in with the N-terminal domain of the these to that the of the hybrid bp and to an for the displaced RNA by in A. that the RNA-DNA hybrid in a T7 is bp J. J. Mol. Biol. PubMed Scopus Google Scholar, A. S. W.T. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, C. C.T. J. Mol. Biol. PubMed Scopus Google and that the RNA is displaced from the hybrid it with the specificity loop and is directed toward a surface of the A. S. W.T. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). with the the region of the RNAP that to the RNA at in the and The for the RNA is with the of RNA cleavage by to acid residues in the N-terminal domain of the RNAP S. PubMed Scopus Google the of the RNA it displaced from the upstream of the RNA-DNA hybrid at A. S. W.T. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). in to the and strands of the DNA at the upstream edge of the DNA nucleotides in the and strands at to a of the (residues that is from the and is by from the N-terminal domain in the of the RNAP must occur during the transition from an IC to an to bring these regions into the of we the of these are to the changes in the IC that must occur during the we the RNA-DNA hybrid observed in the IC by the RNA-DNA hybrid observed in a polymerase complex J. PubMed Scopus Google Scholar). with the cross-linking and in A. S. W.T. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google the of the RNA-DNA hybrid is directed toward the specificity loop and the for the displaced by and PubMed Scopus Google of the RNA-DNA hybrid in in major with the N-terminal region of the RNAP that the changes that occur during the transition to an of the N-terminal domain at a of the region toward the in the specificity loop in the of the specificity loop occur during the of the active of the RNA-DNA hybrid with in the N-terminal of the to bp in with a and and with The of and are of with the in the active site and with the The is that of the T7 RNAP IC PubMed Scopus Google The two bp of the RNA-DNA hybrid observed in the IC are and The RNA of the from the polymerase complex hybrid in the of the in the and are and of T7 RNAP are in and the that in the is in the of The of and T7 RNAP by residues in The and in complex are in and the of is the transition from an to a the and are in a and is of the active site The of and of the and in the complex with that of and the and of T7 RNAP to the of the RNA-DNA the of polymerase that are affected in the region RNAPs that at by OmpT by a number of changes in behavior to the from the the of an RNA-DNA of halted elongation and to at W.T. J. Mol. Biol. PubMed Scopus Google Scholar, R.A. Richardson C.C. J. Biol. PubMed Google Scholar, W.T. J. Mol. Biol. PubMed Scopus Google Scholar, A. W.T. J. Biol. PubMed Scopus Google Scholar, W.T. J. Mol. Biol. 1997; PubMed Scopus Google Scholar, W.T. J. Mol. Biol. PubMed Scopus Google Scholar, W.T. J. Mol. Biol. PubMed Scopus Google Scholar, W.T. J. Mol. Biol. PubMed Scopus Google Scholar). of these are with the that region of the RNAP interacts with the upstream edge of the to the in RNA at of the and RNA A. W.T. J. Biol. PubMed Scopus Google Scholar, J. J. PubMed Scopus Google Scholar, C. J. Biol. Scopus Google the that polymerases is in region at these is with the of in the the that the mutant RNAPs are stable halted and are to changes in template that RNA to that are in the and the W.T. J. Mol. Biol. PubMed Scopus Google W.T. J. Mol. Biol. PubMed Scopus Google of the RNA-DNA hybrid is that the (at a that and into the and at the of the of at the of in a mutant RNAP that at W.T. J. Mol. Biol. 1997; PubMed Scopus Google and it that region of the RNAP in the transition from an IC to an by the of an RNA-DNA hybrid of W.T. J. Mol. Biol. 1997; PubMed Scopus Google Scholar). of residues in the and at the of the in at the of interactions with the domain the a of bp J. Biol. PubMed Scopus Google a variety of of that stages during the transition from to from to and a from to we that of the the with the in a with the loop (at is that that occur in the transition process with the with the displaced RNA the of the by DNA the and initiation regions of the promoter by the of the the release of abortive products changes the in the is in the the release of abortive products J. PubMed Scopus Google C.T. W.T. J. Mol. Biol. Scopus Google Scholar). in the at the of the RNA in a halted forms a with the residues with the of the T7 RNAP is to the at the of the (in the that the of region of the active site is in RNA-DNA hybrid in and is from a RNAP complex that is in the of the the of the active of hybrid into the IC in a and the in the site observed by and PubMed Scopus Google an the the in the into the site with a T7 of of S. J. PubMed Scopus Google observed of is to in T7 and the and are to the and of T7 during the transition from an to a the complex (in the is in a observed to on the template at the of the and to the active the transition to the the to with the template As in the of in the T7 RNAP IC is to that of in complex in the to that the T7 RNAP IC complex by and PubMed Scopus Google a complex that is in the the site is by the and to that of and of during the and in the transition from an to for Ref. Scopus Google Scholar). and PubMed Scopus Google that in of the IC in the for in the in with and in it that is in the of J. PubMed Scopus Google that a in of the from a T7 complex into the IC PubMed Scopus Google is in with Biochemistry. PubMed Scopus Google of these that the transition from an IC to an by T7 RNAP is a complex process that and major of the in the N-terminal it is that to to a of the and to to the RNAPs all of the in the process the complex the of these of in and in Like all RNA polymerases (RNAPs),1 T7 RNAP forms an unstable initiation complex (IC) that synthesizes and releases short abortive products before clearing the promoter and forming a stable elongation complex (EC) (Ref. 1Martin C.T. Muller D.K. Coleman J.E. Biochemistry. 1988; 27: 3966-3974Crossref PubMed Scopus (235) Google Scholar; for review, see Ref. 2McAllister W.T. Nucleic Acids Mol. Biol. 1997; 11: 15-25Crossref Google Scholar). The transition is accompanied by release of promoter contacts, changes in the size of the footprint of the polymerase on the DNA, and changes in accessibility to cleavage by a variety of proteases (3Ikeda R.A. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 3614-3618Crossref PubMed Scopus (147) Google Scholar, 4Place C. Oddos J. Buc H. W.T. Biochemistry. Scopus Google Scholar, J. PubMed Scopus Google Scholar, J. Mol. Biol. PubMed Scopus Google Scholar). these changes that in the of the complex in the of the occur during the and the behavior of mutant and the of changes in template on abortive that during the that from to and a from ∼9 to J. PubMed Scopus Google J. Mol. Biol. 1997; PubMed Scopus Google Scholar, Biochemistry. 1997; PubMed Scopus Google Scholar, W.T. J. Mol. Biol. PubMed Scopus Google Scholar, W.T. J. Mol. Biol. PubMed Scopus Google Scholar, C.T. W.T. J. Mol. Biol. Scopus Google Scholar, C. C.T. J. Biol. PubMed Scopus Google Scholar). the stages to the upstream region of the promoter and the RNAP are the active site the RNA-DNA hybrid a of bp J. PubMed Scopus Google Scholar, C. C.T. J. Biol. PubMed Scopus Google J. J. Mol. Biol. PubMed Scopus Google the of the hybrid to to bp before the to a hybrid of is observed in the C. C.T. J. Biol. PubMed Scopus Google Scholar). cross-linking that stages during promoter at and are before the at C. Oddos J. Buc H. W.T. Biochemistry. Scopus Google Scholar). the the RNA-DNA hybrid is bp and is in a of ∼9 bp J. J. Mol. Biol. PubMed Scopus Google Scholar, A. S. W.T. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, C. C.T. J. Mol. Biol. PubMed Scopus Google Scholar). The upstream of the is to the at the RNA is displaced from the and the is to the of the RNA C. C.T. J. Mol. Biol. PubMed Scopus Google Scholar). As the RNA is displaced from the hybrid at it with a region of the RNAP specificity that is in promoter at the of the RNA from of interactions with the RNAP J. J. Mol. Biol. PubMed Scopus Google Scholar, A. S. W.T. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). for for RNAP with a of for a and for an IC that the in the template PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). for an for that during the transition from an IC to an EC. we the of the T7 RNAP the of nucleic acid-protein cross-linking The are with of the and that major the N-terminal domain occur during the transition to an EC. a number of of T7 RNAP the of a T7 RNAP EC. The in in the of the by nucleic cross-linking are with the of a T7 RNAP IC and that major conformational changes must occur during the transition from an IC to an and PubMed Scopus Google the of a T7 RNAP IC in the of the template the upstream promoter that observed in the T7 complex (in the of are the active site the in of the of the into a that the with to the release of abortive products and promoter during the of J. PubMed Scopus Google C.T. W.T. J. Mol. Biol. Scopus Google Scholar, PubMed Scopus Google the of the and PubMed Scopus Google that of the RNA-DNA hybrid in with the N-terminal domain of the these to that the of the hybrid bp and to an for the displaced RNA by in A. that the RNA-DNA hybrid in a T7 is bp J. J. Mol. Biol. PubMed Scopus Google Scholar, A. S. W.T. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, C. C.T. J. Mol. Biol. PubMed Scopus Google and that the RNA is displaced from the hybrid it with the specificity loop and is directed toward a surface of the A. S. W.T. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). with the the region of the RNAP that to the RNA at in the and The for the RNA is with the of RNA cleavage by to acid residues in the N-terminal domain of the RNAP S. PubMed Scopus Google the of the RNA it displaced from the upstream of the RNA-DNA hybrid at A. S. W.T. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). in to the and strands of the DNA at the upstream edge of the DNA nucleotides in the and strands at to a of the (residues that is from the and is by from the N-terminal domain in the of the RNAP must occur during the transition from an IC to an to bring these regions into the of we the of these are to the changes in the IC that must occur during the we the RNA-DNA hybrid observed in the IC by the RNA-DNA hybrid observed in a polymerase complex J. PubMed Scopus Google Scholar). with the cross-linking and in A. S. W.T. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google the of the RNA-DNA hybrid is directed toward the specificity loop and the for the displaced by and PubMed Scopus Google of the RNA-DNA hybrid in in major with the N-terminal region of the RNAP that the changes that occur during the transition to an of the N-terminal domain at a of the region toward the in the specificity loop in the of the specificity loop occur during the of polymerase that are affected in the region RNAPs that at by OmpT by a number of changes in behavior to the from the the of an RNA-DNA of halted elongation and to at W.T. J. Mol. Biol. PubMed Scopus Google Scholar, R.A. Richardson C.C. J. Biol. PubMed Google Scholar, W.T. J. Mol. Biol. PubMed Scopus Google Scholar, A. W.T. J. Biol. PubMed Scopus Google Scholar, W.T. J. Mol. Biol. 1997; PubMed Scopus Google Scholar, W.T. J. Mol. Biol. PubMed Scopus Google Scholar, W.T. J. Mol. Biol. PubMed Scopus Google Scholar, W.T. J. Mol. Biol. PubMed Scopus Google Scholar). of these are with the that region of the RNAP interacts with the upstream edge of the to the in RNA at of the and RNA A. W.T. J. Biol. PubMed Scopus Google Scholar, J. J. PubMed Scopus Google Scholar, C. J. Biol. Scopus Google the that polymerases is in region at these is with the of in the the that the mutant RNAPs are stable halted and are to changes in template that RNA to that are in the and the W.T. J. Mol. Biol. PubMed Scopus Google W.T. J. Mol. Biol. PubMed Scopus Google of the RNA-DNA hybrid is that the (at a that and into the and at the of the of at the of in a mutant RNAP that at W.T. J. Mol. Biol. 1997; PubMed Scopus Google and it that region of the RNAP in the transition from an IC to an by the of an RNA-DNA hybrid of W.T. J. Mol. Biol. 1997; PubMed Scopus Google Scholar). of residues in the and at the of the in at the of interactions with the domain the a of bp J. Biol. PubMed Scopus Google a variety of of that stages during the transition from to from to and a from to we that of the the with the in a with the loop (at is that that occur in the transition process with the with the displaced RNA the of the by DNA the and initiation regions of the promoter by the of the the release of abortive products changes the in the is in the the release of abortive products J. PubMed Scopus Google C.T. W.T. J. Mol. Biol. Scopus Google Scholar). in the at the of the RNA in a halted forms a with the residues with the of the T7 RNAP is to the at the of the (in the that the of region of the active site is in RNA-DNA hybrid in and is from a RNAP complex that is in the of the the of the active of hybrid into the IC in a and the in the site observed by and PubMed Scopus Google an the the in the into the site with a T7 of of S. J. PubMed Scopus Google observed of is to in T7 and the and are to the and of T7 during the transition from an to a the complex (in the is in a observed to on the template at the of the and to the active the transition to the the to with the template As in the of in the T7 RNAP IC is to that of in complex in the to that the T7 RNAP IC complex by and PubMed Scopus Google a complex that is in the the site is by the and to that of and of during the and in the transition from an to for Ref. Scopus Google Scholar). and PubMed Scopus Google that in of the IC in the for in the in with and in it that is in the of J. PubMed Scopus Google that a in of the from a T7 complex into the IC PubMed Scopus Google is in with Biochemistry. PubMed Scopus Google a number of of T7 RNAP the of a T7 RNAP EC. The in in the of the by nucleic cross-linking are with the of a T7 RNAP IC and that major conformational changes must occur during the transition from an IC to an EC. and PubMed Scopus Google the of a T7 RNAP IC in the of the template the upstream promoter that observed in the T7 complex (in the of are the active site the in of the of the into a that the with to the release of abortive products and promoter during the of J. PubMed Scopus Google C.T. W.T. J. Mol. Biol. Scopus Google Scholar, PubMed Scopus Google Scholar). the of the and PubMed Scopus Google that of the RNA-DNA hybrid in with the N-terminal domain of the these to that the of the hybrid bp and to an for the displaced RNA by in A. that the RNA-DNA hybrid in a T7 is bp J. J. Mol. Biol. PubMed Scopus Google Scholar, A. S. W.T. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, C. C.T. J. Mol. Biol. PubMed Scopus Google and that the RNA is displaced from the hybrid it with the specificity loop and is directed toward a surface of the A. S. W.T. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). with the the region of the RNAP that to the RNA at in the and The for the RNA is with the of RNA cleavage by to acid residues in the N-terminal domain of the RNAP S. PubMed Scopus Google Scholar). The the of the RNA it displaced from the upstream of the RNA-DNA hybrid at A. S. W.T. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). in to the and strands of the DNA at the upstream edge of the DNA nucleotides in the and strands at to a of the (residues that is from the and is by from the N-terminal domain in the of the RNAP must occur during the transition from an IC to an to bring these regions into the of we the of these are to the changes in the IC that must occur during the we the RNA-DNA hybrid observed in the IC by the RNA-DNA hybrid observed in a polymerase complex J. PubMed Scopus Google Scholar). with the cross-linking and in A. S. W.T. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google the of the RNA-DNA hybrid is directed toward the specificity loop and the for the displaced by and PubMed Scopus Google of the RNA-DNA hybrid in in major with the N-terminal region of the RNAP that the changes that occur during the transition to an of the N-terminal domain at a of the region toward the in the specificity loop in the of the specificity loop occur during the of polymerase that are affected in the region RNAPs that at by OmpT by a number of changes in behavior to the from the the of an RNA-DNA of halted elongation and to at W.T. J. Mol. Biol. PubMed Scopus Google Scholar, R.A. Richardson C.C. J. Biol. PubMed Google Scholar, W.T. J. Mol. Biol. PubMed Scopus Google Scholar, A. W.T. J. Biol. PubMed Scopus Google Scholar, W.T. J. Mol. Biol. 1997; PubMed Scopus Google Scholar, W.T. J. Mol. Biol. PubMed Scopus Google Scholar, W.T. J. Mol. Biol. PubMed Scopus Google Scholar, W.T. J. Mol. Biol. PubMed Scopus Google Scholar). of these are with the that region of the RNAP interacts with the upstream edge of the to the in RNA at of the and RNA A. W.T. J. Biol. PubMed Scopus Google Scholar, J. J. PubMed Scopus Google Scholar, C. J. Biol. Scopus Google the that polymerases is in region at these is with the of in the the that the mutant RNAPs are stable halted and are to changes in template that RNA to that are in the and the W.T. J. Mol. Biol. PubMed Scopus Google W.T. J. Mol. Biol. PubMed Scopus Google Scholar). The of the RNA-DNA hybrid is that the (at a that and into the and at the of the of at the of in a mutant RNAP that at W.T. J. Mol. Biol. 1997; PubMed Scopus Google and it that region of the RNAP in the transition from an IC to an by the of an RNA-DNA hybrid of W.T. J. Mol. Biol. 1997; PubMed Scopus Google Scholar). of residues in the and at the of the in at the of interactions with the domain the a of bp J. Biol. PubMed Scopus Google Scholar). As a variety of of that stages during the transition from to from to and a from to we that of the the with the in a with the loop (at is that that occur in the transition process with the with the displaced RNA the of the by DNA the and initiation regions of the promoter by the of the the release of abortive products changes the in the is in the the release of abortive products J. PubMed Scopus Google C.T. W.T. J. Mol. Biol. Scopus Google Scholar). As in the at the of the RNA in a halted forms a with the residues with the of the T7 RNAP is to the at the of the (in the that the of region of the active site is in The RNA-DNA hybrid in and is from a RNAP complex that is in the of the the of the active of hybrid into the IC in a and the in the site observed by and PubMed Scopus Google an the the in the into the site with a T7 of of S. J. PubMed Scopus Google observed of is to in T7 and the and are to the and of T7 during the transition from an to a the complex (in the is in a observed to on the template at the of the and to the active the transition to the the to with the template As in the of in the T7 RNAP IC is to that of in complex in the to that the T7 RNAP IC complex by and PubMed Scopus Google a complex that is in the the site is by the and to that of and of during the and in the transition from an to for Ref. Scopus Google Scholar). and PubMed Scopus Google that in of the IC in the for in the in with and in it that is in the of J. PubMed Scopus Google that a in of the from a T7 complex into the IC PubMed Scopus Google is in with Biochemistry. PubMed Scopus Google of these that the transition from an IC to an by T7 RNAP is a complex process that and major of the in the N-terminal it is that to to a of the and to to the RNAPs all of the in the process the complex the of these of in and in The of these that the transition from an IC to an by T7 RNAP is a complex process that and major of the in the N-terminal it is that to to a of the and to to the RNAPs all of the in the process the complex the of these of in and in

The Drosophila systemic immune response against many Gram-positive bacteria and fungi is mediated by the Toll pathway. How Toll-regulated effectors actually fulfill this role remains poorly understood as the known Toll-regulated antimicrobial peptide (AMP) genes are active only against filamentous fungi and not against Gram-positive bacteria or yeasts. Besides AMPs, two families of peptides secreted in response to infectious stimuli that activate the Toll pathway have been identified, namely Bomanins and peptides derived from a polyprotein precursor known as Baramicin A (BaraA). Unexpectedly, the deletion of a cluster of 10 Bomanins phenocopies the Toll mutant phenotype of susceptibility to infections. Here, we demonstrate that BaraA is required specifically in the host defense against Enterococcus faecalis and against the entomopathogenic fungus Metarhizium robertsii , albeit the fungal burden is not altered in BaraA mutants. BaraA protects the fly from the action of distinct toxins secreted by these Gram-positive and fungal pathogens, respectively, Enterocin V and Destruxin A. The injection of Destruxin A leads to the rapid paralysis of flies, whether wild type (WT) or mutant. However, a larger fraction of wild-type than BaraA flies recovers from paralysis within 5 to 10 h. BaraAs' function in protecting the host from the deleterious action of Destruxin is required in glial cells, highlighting a resilience role for the Toll pathway in the nervous system against microbial virulence factors. Thus, in complement to the current paradigm, innate immunity can cope effectively with the effects of toxins secreted by pathogens through the secretion of dedicated peptides, independently of xenobiotics detoxification pathways.