Transcription Initiation at the TATA-less Spliced Leader RNA Gene Promoter Requires at Least Two DNA-binding Proteins and a Tripartite Architecture That Includes an Initiator Element

Abstract

Eukaryotic transcriptional regulatory signals, defined as core and activator promoter elements, have yet to be identified in the earliest diverging group of eukaryotes, the primitive protozoans, which include the Trypanosomatidae family of parasites. The divergence within this family is highlighted by the apparent absence of the “universal” transcription factor TATA-binding protein. To understand gene expression in these protists, we have investigated spliced leader RNA gene transcription. The RNA product of this gene provides an m7G cap and a 39-nucleotide leader sequence to all cellular mRNAs via a trans-splicing reaction. Regulation of spliced leader RNA synthesis is controlled by a tripartite promoter located exclusively upstream from the transcription start site. Proteins PBP-1 and PBP-2 bind to two of the three promoter elements in the trypanosomatid Leptomonas seymouri. They represent the first trypanosome transcription factors with typical double-stranded DNA binding site recognition. These proteins ensure efficient transcription. However, accurate initiation is determined an initiator element with a a loose consensus of CYAC/AYR (+1), which differs from that found in metazoan initiator elements as well as from that identified in one of the earliest diverging protozoans, Trichomonas vaginalis. Trypanosomes may utilize initiator element-protein interactions, and not TATA sequence–TATA-binding protein interactions, to direct proper transcription initiation by RNA polymerase II. Eukaryotic transcriptional regulatory signals, defined as core and activator promoter elements, have yet to be identified in the earliest diverging group of eukaryotes, the primitive protozoans, which include the Trypanosomatidae family of parasites. The divergence within this family is highlighted by the apparent absence of the “universal” transcription factor TATA-binding protein. To understand gene expression in these protists, we have investigated spliced leader RNA gene transcription. The RNA product of this gene provides an m7G cap and a 39-nucleotide leader sequence to all cellular mRNAs via a trans-splicing reaction. Regulation of spliced leader RNA synthesis is controlled by a tripartite promoter located exclusively upstream from the transcription start site. Proteins PBP-1 and PBP-2 bind to two of the three promoter elements in the trypanosomatid Leptomonas seymouri. They represent the first trypanosome transcription factors with typical double-stranded DNA binding site recognition. These proteins ensure efficient transcription. However, accurate initiation is determined an initiator element with a a loose consensus of CYAC/AYR (+1), which differs from that found in metazoan initiator elements as well as from that identified in one of the earliest diverging protozoans, Trichomonas vaginalis. Trypanosomes may utilize initiator element-protein interactions, and not TATA sequence–TATA-binding protein interactions, to direct proper transcription initiation by RNA polymerase II. spliced leader nucleotide(s) polymerase base pair(s) small nuclear initiator trypanosome initiator wild type polymerase chain reaction pBluescript SK II Molecular studies of trypanosomatids, a ubiquitous and diverse family of protozoan pathogens, have revealed strikingly unusual mechanisms of mRNA synthesis. One central device is that two independent transcription events direct each mRNA produced in the trypanosome nucleus (for review, see Ref. 1Vanhamme L. Pays E. Microbiol. Rev. 1995; 59: 223-240Crossref PubMed Google Scholar). The protein-coding portion is transcribed as a single primary mRNA, often containing several open reading frames flanked by 5′- and 3′-untranslated regions. The capped 5′-end portion is transcribed as a short spliced leader (SL)1 RNA. The two parts are fused in a trans-splicing reaction that yields a functional mRNA. During fusion, the 39 nt present on the 5′-end of the SL RNA (and referred to as the SL) are transferred to a region upstream from the coding region on the primary mRNA (2Pays E. Broda P.M. Olvier S.G. Sims P. The Eukaryotic Microbial Genome. Cambridge University Press, Cambridge1993: 99-132Google Scholar). Addition of the SL provides each mRNA with an m7G cap as well as four extensively methylated nucleotides, at positions 1–4 within the 39-nt SL RNA (3Bangs J.D. Crain P.F. Hashizume T. McCloskey J.A. Boothroyd J.C. J. Biol. Chem. 1992; 267: 9805-9815Abstract Full Text PDF PubMed Google Scholar). The SL RNA is transcribed from a highly reiterated set of genes. In contrast to the long primary transcripts that form the bulk of the mature mRNA, each SL RNA has a discrete transcriptional start site. α-Amanitin studies show that it is very probable, though not proven, that the SL RNA gene is transcribed by RNA polymerase (pol) II. The primary SL RNA transcript and the transcript present in the trans-splicing spliceosome possess identical 5′- and 3′-ends, indicating that both transcription initiation and termination regulate the accumulation of SL RNA. SL RNA expression has been monitored using independent, tagged gene copies positioned on selectable shuttle vectors that are stably maintained in various trypanosomatids (4Günzl A. Tschudi C. Nakaar V. Ullu E. J. Biol. Chem. 1995; 270: 17287-17291Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 5Yu M.C. Sturm N. Saito R. Roberts T. Campbell D. Mol. Biochem. Parasitol. 1998; 94: 265-281Crossref PubMed Scopus (27) Google Scholar, 6Agami R. Aly R. Halman S. Shapira M. Nucleic Acids Res. 1994; 22: 1959-1965Crossref PubMed Scopus (42) Google Scholar). In the simple trypanosomatid Leptomonas seymouri, a 95-bp region upstream of the SL RNA intragenic region followed by 70 bp of downstream sequence is sufficient to produce properly initiated and terminated SL RNA (7Hartree D. Bellofatto V. Mol. Biochem. Parasitol. 1995; 71: 27-39Crossref PubMed Scopus (34) Google Scholar). These results have been recapitulated in vitro using homologous parasite nuclear extracts (8Huie J. He P. Bellofatto V. Mol. Biochem. Parasitol. 1997; 90: 183-192Crossref PubMed Scopus (23) Google Scholar). Unusual promoter architecture in this group of primitive eukaryotes, compared with typical metazoans, appears to be the rule. The U6 small nuclear (sn) RNA gene promoter contains three elements: one located within the 5′-portion of the intragenic region and two located within an upstream, but inversely oriented, tRNA gene. The two intragenic tRNA promoter elements, called A and B boxes, cofunction in both U6 and tRNA expression (9Nakaar V. Dare A.O. Hong D. Ullu E. Tschudi C. Mol. Cell. Biol. 1994; 14: 6736-6742Crossref PubMed Scopus (75) Google Scholar). Two abundant cell surface proteins in the African trypanosome Trypanosoma brucei are encoded by genes with promoter elements that resemble RNA pol I promoters in both structure and α-amanitin resistance. Aside from these two protein coding genes in T. brucei, all other trypanosomatid mRNAs are α-amanitin-sensitive and thus transcribed by RNA pol II (for review, see Refs. 10Graham S.V. Parasitol. Today. 1995; 11: 217-223Abstract Full Text PDF PubMed Scopus (69) Google Scholar and 11Cross G. Wirtz L. Navarro M. Mol. Biochem. Parasitol. 1998; 91: 77-91Crossref PubMed Scopus (79) Google Scholar). Transcriptional start sites for primary mRNAs have been extremely difficult to detect. Two putative promoter regions were tentatively defined as transcriptionally void regions upstream from the highly transcribed actin and HSP 70 genes (12Ben Amar M. Jefferies D. Pays A. Bakalara N. Kendall G. Pays E. Nucleic Acids Res. 1991; 19: 5857-5862Crossref PubMed Scopus (31) Google Scholar, 13Lee M.G. Mol. Cell. Biol. 1996; 16: 1220-1230Crossref PubMed Scopus (47) Google Scholar). However, placement of these sequences upstream from a luciferase coding region did not yield even modest levels of reporter gene activity (14McAndrew M. Graham S. Hartmann C. Clayton C. Exp. Parasitol. 1998; 90: 65-76Crossref PubMed Scopus (40) Google Scholar). Moreover, in the absence of any putative trypanosome promoter regions, Escherichia coli pBR 322-derived sequences drive expression of reporter genes, such as the chloramphenicol acetyltransferase gene. Models to these that RNA pol II may not be to promoter sites to mRNA synthesis. Addition of an SL to these mRNA as mRNAs mature present a transcriptional of the SL RNA gene promoter using an in vitro transcription that in transcription. In a of any defined trypanosome transcription PBP-1 and PBP-2 Bellofatto V. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google which are proteins identified in as the first proteins that to efficient SL RNA transcription. These studies that 5′-end of the SL which is to proper of the is on the of a the trypanosome initiator we for a factor for transcription which the L. V. Nucleic Acids Res. 16: PubMed Scopus Google at to were by and with (7Hartree D. Bellofatto V. Mol. Biochem. Parasitol. 1995; 71: 27-39Crossref PubMed Scopus (34) Google Scholar). of the were at The cell in cell of containing on were A the nuclear in cell of at and cell of The by The using of to the The by and with of the with at and this The by and were at The nuclear protein The by the nuclear of at and a as Bellofatto V. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). Proteins were using a in The for transcription. using the for the nuclear The protein of The DNA PBP-1 and PBP-2 proteins were as Bellofatto V. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google and at protein to the transcription reaction. the the from In all SL RNA a present in the coding region and The sequence of the to nt of the upstream region of the SL RNA gene has been (7Hartree D. Bellofatto V. Mol. Biochem. Parasitol. 1995; 71: 27-39Crossref PubMed Scopus (34) Google Scholar). In each the were The DNA type and were from using and to the nt upstream region of the and the nt downstream region of the SL RNA other were by DNA to base sequences are in and of the start site is determined by the is the and of the start sites that were identified in the transcription The nt that each start site are a start site. The nt The transcribed sequence is in and the sequences are on the start were double-stranded a a and a a DNA which the nt promoter region of SL RNA gene by of pBluescript SK II region sequence The first three were by The by using the as and SK and from The U6 gene tagged by of a sequence nt by using the U6 gene from and the and the sequence is to the to nt region and the the sequence is and to the to nt sequence of the U6 gene. The product by and the two and the product with the and tagged U6 gene and referred to as The were in in a containing of nuclear of DNA of of and The were for at and terminated by of and of with of The RNA with The in of To PBP-1 and PBP-2 a of bp of to the to the were using and from RNA and 70 of were and and to of and the at for The were in and to a To ensure that of in transcription and not an to each reaction. the tagged U6 gene in each SL RNA transcription reaction. this gene the as the SL RNA gene the U6 and SL RNA transcripts were and transcribed using the The in the U6 gene in a such that the product from the U6 gene nt that from the SL RNA gene. product by of The of the from the U6 and SL RNA product set as The of product from each SL RNA gene compared with the product within each reaction and as a to SL RNA transcription. The for the To a DNA for the of the a using the which contains the SL RNA and two SL The at nt of the SL RNA the at nt and has the promoter at 5′-end to for by in with RNA transcription produced a that nt of the in vitro transcribed tagged SL RNA and nt of the SL RNA. in vitro were as (8Huie J. He P. Bellofatto V. Mol. Biochem. Parasitol. 1997; 90: 183-192Crossref PubMed Scopus (23) Google and RNA and in of A the with of The were at regions were using and and the RNA with and in were on a in and results were using of a gene promoter in these have that the SL RNA gene promoter within the nt upstream of the transcription start site R. Aly R. Halman S. Shapira M. Nucleic Acids Res. 1994; 22: 1959-1965Crossref PubMed Scopus (42) Google D. Bellofatto V. Mol. Biochem. Parasitol. 1995; 71: 27-39Crossref PubMed Scopus (34) Google Scholar, A. Ullu E. M. S. J. E. S. S. Dare A. Tschudi C. Mol. Biochem. Parasitol. 1997; PubMed Scopus Google Scholar, M.G. Campbell J. 1994; PubMed Scopus Google Scholar). In promoter has revealed a tripartite promoter architecture (7Hartree D. Bellofatto V. Mol. Biochem. Parasitol. 1995; 71: 27-39Crossref PubMed Scopus (34) Google Scholar, A. Ullu E. M. S. J. E. S. S. Dare A. Tschudi C. Mol. Biochem. Parasitol. 1997; PubMed Scopus Google Scholar). However, the of in is that promoter that produced initiated SL which not be from that transcription. To the each promoter element to the transcriptional homologous nuclear extracts were produced which transcription on DNA containing the upstream nt to a coding sequence (8Huie J. He P. Bellofatto V. Mol. Biochem. Parasitol. 1997; 90: 183-192Crossref PubMed Scopus (23) Google Scholar). The L. in vitro transcription extracts and thus transcription of other nuclear In a of these the SL RNA gene a that is transcribed as of the SL RNA and Ref. J. He P. Bellofatto V. Mol. Biochem. Parasitol. 1997; 90: 183-192Crossref PubMed Scopus (23) Google Scholar). are by using the of the sequence as the that the SL RNA gene vitro to produce an initiated SL RNA. transcriptional of the revealed that the two upstream elements and of the core SL RNA gene promoter were for efficient transcription. However, these did not RNA start site and an gene promoters in and the SL RNA gene promoter have that PBP-1 and PBP-2 be for transcriptional start sites and Press, Scholar). a of the core which at proper transcription initiation The in been a of SL RNA. the of initiated SL in to produce a for the in the produced transcripts that initiated at we it to been on in vitro transcripts using an that SL RNA sequences from nt to nt The of in upstream of the at nt the SL present within the nuclear a of nt terminated in SL a of the of the nt The RNA in B that the of the SL transcribed from a SL RNA gene properly in the of the SL RNA transcribed from the DNA of SL by is with this (7Hartree D. Bellofatto V. Mol. Biochem. Parasitol. 1995; 71: 27-39Crossref PubMed Scopus (34) Google Scholar). The of the product in the compared with the with the in transcription levels from the The the that of RNA were in each These results the for the nt region exclusively in transcription start site and not in transcription termination of the SL RNA gene. In the absence of a TATA to direct start site in any of the trypanosome gene we that element within the tripartite promoter of the SL RNA gene to accurate RNA initiation L. D. Rev. Biochem. 1995; PubMed Scopus Google Scholar). A of and genes on an often a TATA for proper initiation 1997; PubMed Scopus Google Scholar, S. D. Cell. Full Text PDF PubMed Scopus Google Scholar, R. G. J. 1992; 11: PubMed Scopus Google Scholar, E. R. Mol. Cell. Biol. 1991; 11: PubMed Scopus Google Scholar, E. J. Nucleic Acids Res. Scopus Google Scholar). a of the in the element functional to the metazoan and is referred to as a However, a sequence the metazoan and a In metazoans, a loose consensus of in which it is for the to be an A for activity S. 1996; PubMed Scopus Google Scholar). A base of the sequence that SL RNA transcription start sites in Trypanosomatidae a consensus in which the is the found in is in of this trypanosome and metazoan it is to the trypanosome as To the of the were in and the nt region of the SL RNA each of the nt region and were to nuclear the transcription of the nt region of the transcripts to at the A of the sequence with the consensus that a been by the of the nt region the sequence from which is been with In the a at to SL RNA transcription. of the nt region and synthesis of properly initiated SL of the sequence with In the absence of a in the by the nucleotides, were by the transcription initiated at several with These initiation sites were as located at to the start located at located at located at In the of the start site with the of the in this by an that is identical to that within the The start site in the of RNA with modest in the of RNA start site is by a sequence that is identical to the region that the is an A in of a and the that is as the initiation is in of A. of at three nt within the nt upstream of the start site is for In the at positions and nt were from to on start site did this a these show that the is to the nt to the Transcriptional of SL RNA genes in three trypanosomatids have that within the SL RNA sequence on both transcription and start site these were not in M.C. Sturm N. Saito R. Roberts T. Campbell D. Mol. Biochem. Parasitol. 1998; 94: 265-281Crossref PubMed Scopus (27) Google Scholar, A. Ullu E. M. S. J. E. S. S. Dare A. Tschudi C. Mol. Biochem. Parasitol. 1997; PubMed Scopus Google Bellofatto V. Parasitol. Res. PubMed Scopus Google Scholar). an of we determined the SL RNA sequence the Moreover, by the of four we the of the SL RNA sequence to the upstream that the upstream and intragenic regions be in this The did not start site In of the coding region with sequences did not the start not These results that the downstream of the the In both the and the nt upstream from the start site were followed by an which is but not identical to the The of the A to at that this present in SL RNA sequences all trypanosomatid is not for In both and gene sequence elements to with the DNA binding of one proteins to form a These are often to the each To the within the SL RNA gene we the and by levels were by and to the of U6 transcription which in each reaction not of that the and by nt the of transcription and the two elements on the of the DNA which by the of to activity A in the and a on expression levels These that and to efficient SL RNA To the and regions, we these two promoter elements A and see show that an in the the and the a in transcription levels and levels the from the and regions, the not by the transcriptional for transcription initiation any two of the and elements it is that SL RNA expression that the three elements as a to regulate gene the trypanosome SL RNA gene an that in mRNA it is that SL RNA transcription may of the that to the transcription of genes in other J. Mol. Cell. Biol. 1996; 16: PubMed Google Scholar, Nucleic Acids Res. 1998; PubMed Scopus Google Scholar, Mol. Cell. Biol. 1996; 16: PubMed Google Scholar, M. R. R. N. P. M. N. Mol. Cell. Biol. 1998; PubMed Google Scholar). In the the two promoter elements, the and TATA within the gene promoters RNA pol II is to the S. C. D. J. Mol. Cell. Biol. 1994; 14: PubMed Scopus Google Scholar). To this the in trypanosome SL RNA that sequences the and regions, the and regions, were for RNA polymerase type SL RNA gene promoters are α-amanitin-sensitive in in studies and thus RNA pol II for transcription. The were in to α-amanitin compared with the transcriptional levels in each are not the of an RNA polymerase within the initiation The for and to be within nt of each other is with the in which PBP-1 and PBP-2 were to form at the SL RNA gene promoter Bellofatto V. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). To the of PBP-1 and PBP-2 in SL RNA transcription we protein and using in vitro transcription that transcriptionally nuclear extracts PBP-1 and PBP-2 not that of PBP-1 and PBP-2 proteins by the of of binding sites a DNA transcription. studies using of the PBP-1 PBP-2 binding sites transcription efficient in expression which is with that PBP-2 DNA Bellofatto V. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). the region to the transcription reaction transcription from that a protein is The transcriptional of each of the three SL RNA gene promoter elements in transcription not by the of a DNA To the for PBP-1 and PBP-2 in SL RNA we a nuclear that transcriptionally but from the in that it of PBP-1 and of the PBP-1 and PBP-2 protein using DNA transcription Transcriptional activity highly PBP-1 and PBP-2 were to the reaction and of did not activity of activity not to be using highly protein is often in PBP-1 and PBP-2 bind to the and elements in and these two proteins transcription in in vitro transcription it is that PBP-1 and PBP-2 as factors in SL RNA In the of RNA often promoters in the absence of proteins in this is in contrast to and metazoan RNA trypanosomatids are of an these that the of gene promoters by transcription factors in contrast to RNA polymerase is an of the SL RNA gene promoters from the trypanosomatids in which the are has revealed a highly at the transcriptional start site. In the in vitro transcription that the L. sequence sites as of these initiation sites to the regions of the for initiator of as well as of the followed by a for initiator The of sites the from the region provides with the of sites that an at positions that a is not for However, the of three of nt followed by a at in the the at is may be for DNA and the for the upstream sites in the nt is a in the of all the trypanosomatid SL RNA gene revealed that an A at nt to each start site for that are an A is present at the nt well in both the identified by and the elements in the trypanosomatid the and nt positions were in the start sites and this in the start is identical to that found in metazoan The trypanosome sequence is to a metazoan element it to direct transcription The metazoan consensus sequence is defined as which is but from the trypanosome of the region from a protozoan even Trichomonas has an element that contains a highly D. P. Mol. Cell. Biol. 19: PubMed Scopus Google Scholar). the T. and trypanosome a with the metazoan are from each is that start sites in each and nt from the upstream The DNA by and C. J. PubMed Scopus Google to start site by RNA pol II transcription of mRNA coding genes in may be to this promoter with RNA polymerase the at a downstream from the TATA However, in the two genes compared in the transcription initiation within a from the TATA in the and gene To for this RNA pol II is to the DNA and the start site by downstream of the an sequence within the and transcription. The trypanosome are with an RNA in these the of a consensus start sites the region and the and the RNA polymerase to transcripts at the region and the Moreover, appears to be to a set of nt downstream from the In the of the of the initiation to at sequences that were within a from the and nt consensus the located of this to be In the of the the site that it the of the polymerase RNA polymerase located the with to the CYAC/AYR consensus but to the in a DNA in a that the of DNA a protein in A. 1998; PubMed Scopus Google Scholar). These the for RNA polymerase for initiation sites within transcriptional The SL RNA gene promoter is containing three elements that all within the upstream bp of the region M.C. Sturm N. Saito R. Roberts T. Campbell D. Mol. Biochem. Parasitol. 1998; 94: 265-281Crossref PubMed Scopus (27) Google Scholar, D. Bellofatto V. Mol. Biochem. Parasitol. 1995; 71: 27-39Crossref PubMed Scopus (34) Google Scholar, A. Ullu E. M. S. J. E. S. S. Dare A. Tschudi C. Mol. Biochem. Parasitol. 1997; PubMed Scopus Google Scholar). Two of these elements with In the of the two elements, two transcription factors have been identified and to be for transcription in for a transcription which with the is by the promoter structure and the of two protein are from the promoters found in other In the of genes in and metazoans, a single sequence element element is by a protein to the that RNA pol II transcription. it is RNA pol II the SL it the SL RNA promoter architecture is for this of genes. The of the three promoter elements that a of the and regions results in a in transcription and of the region the binding of PBP-1 and PBP-2 to DNA as by Bellofatto V. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). Moreover, of PBP-1 and PBP-2 from extracts RNA and proteins the of and is to PBP-1 and PBP-2 to the promoter is that the SL RNA gene is transcribed by RNA pol have not been is on transcription studies using in which the of the SL RNA but not the mRNA RNA pol mRNA gene promoters in trypanosomatids (14McAndrew M. Graham S. Hartmann C. Clayton C. Exp. Parasitol. 1998; 90: 65-76Crossref PubMed Scopus (40) Google Scholar, A. Ullu E. M. S. J. E. S. S. Dare A. Tschudi C. Mol. Biochem. Parasitol. 1997; PubMed Scopus Google Scholar, M.G. Campbell J. 1994; PubMed Scopus Google Scholar, Bellofatto V. Parasitol. Res. PubMed Scopus Google Scholar). The binding of PBP-1 and PBP-2 to two core SL RNA gene promoter elements that a of RNA pol II genes by proteins upstream from a discrete start site. A may not upstream from genes, thus the to discrete start sites for these genes. RNA pol II may be to two very transcription genes and the SL in RNA pol and genes in have promoter and transcription element within a SL RNA gene promoter may be to the of identified that SL RNA genes. transcription initiation of the SL in the SL RNA sequences but not any DNA sequences to the RNA start site J.D. J. PubMed Scopus Google Scholar). defined elements have been identified in and SL RNA genes in it is not yet which promoter sequences are for proper transcription initiation of SL RNA genes in these L. J. C. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). in the SL RNA is extensively a cap structure via the of within both the and base trypanosome SL RNA contains this extensively and capped sequence at of this sequence to transcription initiation the of a functional SL RNA. These and the of an element in initiated and thus functional SL and of the Bellofatto for on this