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
1999; Elsevier BV; Volume: 274; Issue: 45 Linguagem: Inglês
10.1074/jbc.274.45.31947
ISSN1083-351X
AutoresHua Luo, Gwen Gilinger, Devi Mukherjee, Vivian Bellofatto,
Tópico(s)CRISPR and Genetic Engineering
ResumoEukaryotic 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 explain these findings suggest that RNA pol II may not be recruited to specific promoter sites to initiate mRNA synthesis. Addition of an SL to these jagged mRNA 5′-ends would polish them as mRNAs mature into translatable units. We present a detailed transcriptional analysis of the SL RNA gene promoter using an in vitro transcription system that faithfully recapitulates in vivo transcription. In a dearth of any previously defined trypanosome transcription factors, PBP-1 and PBP-2 (15Luo H. Bellofatto V. J. Biol. Chem. 1997; 272: 33344-33352Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), which are sequence-specific DNA-binding proteins initially identified in our laboratory, emerge as the first proteins that function to promote efficient SL RNA transcription. These studies also reveal that correct 5′-end formation of the SL RNA, which is crucial to proper capping of the SL, is dependent on the presence of a 5-bp element, the trypanosome initiator (Inrt). Finally, we provide evidence for a trans-acting factor necessary for transcription which binds the Inrt. L. seymouri (ATCC 30220) was grown inTrypanosoma mega medium (16Bellofatto V. Cross G.A.M. Nucleic Acids Res. 1988; 16: 3455-3469Crossref PubMed Scopus (15) Google Scholar) at 28 °C to log phase. Cells were harvested by centrifugation (3,900 × g, 20 min, 4 °C) and washed twice with buffer 1 (20 mm Tris-HCl, pH 7.4, 100 mm NaCl, 3 mm MgCl2) (7Hartree D. Bellofatto V. Mol. Biochem. Parasitol. 1995; 71: 27-39Crossref PubMed Scopus (34) Google Scholar). All of the following steps were performed at 4 °C. The cell pellet was resuspended in 1 packed cell volume of buffer 2 (10 mmHEPES-KOH, pH 7.9, 150 mm sucrose, 2.5 mmMgCl2, 1 mm EDTA, 2.5 mmdithiothreitol, 1 mm leupeptin) containing 20 mm potassium glutamate. After swelling cells on ice (10 min), they were Dounce homogenized (size A pestle) until disrupted. After centrifugation, the nuclear pellet was resuspended in 5 packed cell volumes of buffer 2 at 20 mm potassium glutamate, and 1 packed cell volume of (NH4)2SO4(4.1 m) was add dropwise. The extract was cleared by ultracentrifugation (100,000 × g, 35 min, 4 °C). The supernatant was fractionated using solid (NH4)2SO4 (0.33 g/ml of solution). 1 n NaOH (0.1 ml/10 g solid (NH4)2SO4) was added to maintain the pH. The precipitate was collected by centrifugation (15,000 ×g, 20 min, 4 °C) and resuspended with 0.10 volume of the high speed supernatant with buffer 2 at 20 mm potassium glutamate and dialyzed against this buffer. The dialysate was cleared by centrifugation (10,000 × g, 20 min, 4 °C), and aliquots were stored at −70 °C. The nuclear extract protein concentration was 5–10 mg/ml. The P400 fraction was prepared by dialyzing the nuclear extract (∼100 mg of protein) against buffer 2 at 50 mm potassium glutamate and loading onto a 5-ml phosphocellulose (P-11, Whatman) column as described (15Luo H. Bellofatto V. J. Biol. Chem. 1997; 272: 33344-33352Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Proteins were eluted using a step gradient (200, 400, 600 mm potassium glutamate) in buffer 2. The P400 fraction alone was competent for transcription. It was concentrated using the same (NH4)2SO4 precipitate method described above for the nuclear extract preparation. The final protein concentration of P400 was ∼5 mg/ml. The specific DNA affinity-purified PBP-1 and PBP-2 proteins were prepared as described previously (15Luo H. Bellofatto V. J. Biol. Chem. 1997; 272: 33344-33352Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) and dialyzed against buffer 2 at 20 mm potassium glutamate (the final protein concentration was ∼1.6 mg/ml) before addition to the transcription reaction. For the sequestration experiments, the acetylated bovine serum albumin was from New England BioLabs, Inc. In all SL RNA gene-containing templates, a tag was present in the coding region between +48 and +69 nt. The sequence of the 10-nt substitutions between −1 to −90 nt of the upstream region of the SL RNA gene has been published (7Hartree D. Bellofatto V. Mol. Biochem. Parasitol. 1995; 71: 27-39Crossref PubMed Scopus (34) Google Scholar). In each case, the substitutions were nucleotide transversions. The linear DNA templates (wild type (wt)) and 10-nt substitution derivatives) were synthesized from their corresponding plasmids using PCR and primers VB 71 (corresponds to the −73/91 nt upstream region of the promoter) and VB 72 (complements the +197/215 nt downstream region of the SL RNA gene). All other mutated linear templates were synthesized by recombinant PCR. Every DNA was sequenced to confirm base alterations. Mutated sequences are shown in Figs. 1 and 5.Figure 5Positioning of the start site is determined by the Inrt element. PBP-2E is boxed. Arrows indicate the position and relative strength of the start sites that were identified in the transcription assays. The 5 nt that precede each start site are underlined. Abroader arrow signifies a stronger start site. The lowercase letters indicate nt mutations. The wt transcribed sequence is in large bold letters, and the sequences are aligned based on the wt start site.View Large Image Figure ViewerDownload Hi-res image Download (PPT) All competitors were double-stranded DNAs. PBP-1E/2E was a 66-nt (−17/82 nt) DNA; PBP-1E was a 42-nt (−41/83 nt) DNA, and PBP-2E was a 34-nt (−17/50 nt) DNA. Inrt was a 66-nt DNA (5′-TCTAGAACTAGTGGATCCGGGCACCTCGAGACCCTACCATCAAGCTTATCGATACCGTCGACCTCG-3′) which included the −1/20 nt promoter region of SL RNA gene surrounded by part of pBluescript SK II (pBS) polylinker region sequence (Stratagene). The first three DNAs were made by annealing complementary oligonucleotides. The Inrt substrate was amplified by PCR using the top strand as template and SK and KS primers from pBS. The U6 snRNA gene was tagged by insertion of a 21-nt sequence between nt +71/72 by using the wt U6 snRNA gene (a gift from Albrecht Bindereif) and the primers VB 195 (−150/169 nt; 5′-CACTCCTACCTGGACTCGAA-3′) and VB 247 (+58/71 nt; the tag sequence is underlined; 5′-GCTTCAGGGATCCAAGTAAGCGCCTTGCGCAGGGAG-3′) to amplify the −169 to +71 nt region and the primers VB 248 (+72/86 nt; the tag sequence is underlined; 5′-GCTTCATTGGATCCCTGAAGCTGATGTCAATCTTCG-3′) and VB 249 (+86/106 nt; 5′-CGGCGAAAAGCTATATCTCTC-3′) to amplify the +72 to +106 nt sequence of the U6 snRNA gene. The recombinant product was obtained by denaturing and annealing the two PCR products and amplifying the 296-nt product with the VB 195 and VB 249 primers. This 296-nt tagged U6 snRNA gene was cloned into theEcoRV-digested pBS and referred to as pHL-U6. The standard reactions were conducted in 50-μl volume in a buffer containing 10–20 μl of nuclear extract (protein concentration was 5–10 mg/ml), 1 pmol of circular plasmid DNA or 2 pmol of linear DNA, 20 mm potassium glutamate, 3 mmMgCl2, 10 mm HEPES-KOH, pH 7.9, 1 mm dithiothreitol, 3% polyethylene glycol 8000, 20 mm creatine phosphate, 0.48 mg/ml creatine kinase, 1 mm leupeptin, 40 units of porcine RNase inhibitor, 2 mm ATP, 0.8 mm CTP, 0.8 mm GTP, and 0.8 mm UTP. The reactions were incubated for 30 min at 28 °C and terminated by adding 400 μl of stop buffer (5 mm HEPES-KOH, pH 7.9, 5 mm EDTA, 0.5% SDS, 0.3m sodium acetate, and 5 μg of tRNA). Protein was extracted with 400 μl of RNase-free phenol/chloroform/isoamyl alcohol (25:24:1). The RNA was precipitated with ethanol. The pellet was resuspended in 20 μl of primer extension buffer (Promega). To sequester PBP-1 and PBP-2 proteins, a 5-fold molar excess of 66 bp of PBP-1E/2E DNA, relative to the template DNA, was added to the P400 fraction. Primer extension reactions were performed using reagents and protocols obtained from Promega. RNA and 70 fmol of 32P-labeled primer VB 207 (5′-GCTTCAGG GATCCAAGTAAGC-3′) were mixed and heated (5 min, 75 °C) and cooled slowly to 40 °C; 1 unit of avian myeloblastosis virus reverse transcriptase was added, and the annealed primer was extended at 40 °C for 30 min. The products were denatured in 90% formamide (90 °C, 10 min) and applied to a 10% polyacrylamide denaturing (7 m urea) gel. To ensure that quantitation of primer extension products reflected true differences in transcription efficiencies and not experimental variation, an internal control was added to each reaction. Specifically, the tagged U6 snRNA gene described above was included in each SL RNA transcription reaction. Because this gene contained the same tag as the SL RNA gene templates, the U6 snRNA and SL RNA transcripts were specifically hybridized and reverse transcribed using the same primer. The tag was inserted in the U6 snRNA gene in a position such that the primer extension product from the U6 snRNA gene was 22 nt longer than that from the SL RNA gene. Each product was quantitated by PhosphorImager analysis of dried gels. The ratio of the primer extension products from the U6 snRNA and wt SL RNA product was set as 1. The amount of primer extension product from each variant SL RNA gene template was compared with the U6-derived product within each reaction and expressed as a number relative to wt SL RNA transcription. The Ambion RPAII kit was used for the RNase protection assay (Ambion). To obtain a DNA template for the production of the specific riboprobe, a PCR was performed using the plasmid pDEH3, which contains the SL RNA gene, and two SL RNA-specific primers. The sense primer, VB 331, starts at nt +38 of the SL RNA gene; the antisense primer, VB 330, starts at nt +196 and has the T7 promoter at its 5′-end to allow for labeling by in vitrotranscription with T7 polymerase. T7 RNA polymerase-dependent transcription produced a [α32P]UTP-labeled 176-nt riboprobe that could protect 81 nt of the in vitro transcribed tagged SL RNA and 51 nt of the endogenous SL RNA. Trypanosome in vitro transcriptions were performed as described (8Huie J. He P. Bellofatto V. Mol. Biochem. Parasitol. 1997; 90: 183-192Crossref PubMed Scopus (23) Google Scholar), and RNA was precipitated and resuspended in 20 μl of solution A (from the Ambion RPAII kit) with 5 × 104 cpm of riboprobe. The RNAs were hybridized overnight at 42 °C, single-stranded regions were digested using RNase T1 and A, and the resultant RNA was precipitated with ethanol and resuspended in formamide loading buffer. Samples were electrophoresed on a 10% polyacrylamide-urea gel in 1 × TBE, and results were visualized using PhosphorImager analysis. Our investigation of a tractable gene promoter in these ancient eukaryotic protists have established that the SL RNA gene promoter lies within the proximal ∼100 nt upstream of the transcription start site (see Fig. 1) (6Agami R. Aly R. Halman S. Shapira M. Nucleic Acids Res. 1994; 22: 1959-1965Crossref PubMed Scopus (42) Google Scholar,7Hartree D. Bellofatto V. Mol. Biochem. Parasitol. 1995; 71: 27-39Crossref PubMed Scopus (34) Google Scholar, 17Günzl A. Ullu E. Dörner M. Fragoso S. Hoffmann K. Milner J. Morita Y. Nguu E. Vanacova S. Wünsch S. Dare A. Kwon H. Tschudi C. Mol. Biochem. Parasitol. 1997; 85: 67-76Crossref PubMed Scopus (71) Google Scholar, 18Saito R.M. Elgort M.G. Campbell D.A. EMBO J. 1994; 13: 5460-5469Crossref PubMed Scopus (58) Google Scholar). In vivo promoter analysis has revealed a tripartite promoter architecture (7Hartree D. Bellofatto V. Mol. Biochem. Parasitol. 1995; 71: 27-39Crossref PubMed Scopus (34) Google Scholar, 17Günzl A. Ullu E. Dörner M. Fragoso S. Hoffmann K. Milner J. Morita Y. Nguu E. Vanacova S. Wünsch S. Dare A. Kwon H. Tschudi C. Mol. Biochem. Parasitol. 1997; 85: 67-76Crossref PubMed Scopus (71) Google Scholar). However, the limiting component of in vivo analyses is that promoter mutations that produced inaccurately initiated SL RNA, which would turn over rapidly, could not be distinguished from mutations that down-regulated transcription. To investigate the role each promoter element contributes to the transcriptional process, homologous nuclear extracts were produced which could initiate transcription accurately on DNA templates containing the upstream proximal 100 nt adjacent to a guanosine-less coding sequence (G-less cassette) (8Huie J. He P. Bellofatto V. Mol. Biochem. Parasitol. 1997; 90: 183-192Crossref PubMed Scopus (23) Google Scholar). The L. seymouri in vitro transcription extracts contain few RNA-processing enzymes and thus directly assay transcription independently of other nuclear activities. In a recent modification of these assays, shown here, the exogenous SL RNA gene template possesses a 19-nt tag that is transcribed as part of the SL RNA (see "Experimental Procedures" and Ref. 8Huie J. He P. Bellofatto V. Mol. Biochem. Parasitol. 1997; 90: 183-192Crossref PubMed Scopus (23) Google Scholar). RNAs are detected by primer extension reactions using the complement of the tag sequence as the radiolabeled primer. Fig. 2 A, lane 1, demonstrates that the SL RNA gene was transcribedin vitro to produce an accurately initiated SL RNA. Detailed transcriptional analysis of mutated templates, drawn schematically below the data, revealed that the two upstream elements (PBP-1E and PBP-2E) of the core SL RNA gene promoter were necessary for efficient transcription. However, these mutations did not effect RNA start site selection (Fig. 2 A, lanes 4, 5,7, and 8). This was surprising because an analogy between snRNA gene promoters in higher eukaryotes and the SL RNA gene promoter would have predicted that PBP-1 and or PBP-2 would be directly responsible for determining transcriptional start sites (19Lobo S.M. Hernandez N.T. Conaway R.C. Conaway J.W. Transcription Mechanisms and Regulation. Raven Press, New York1994: 127-159Google Scholar). Unexpectedly, a 10-nt mutation of the third core element, which resides at −1/10 nt, completely abolished proper transcription initiation (lane 2). The in vivo phenotype had been a loss of detectable SL RNA. Clearly, the effect of improperly initiated SL RNAs resulted in their rapid turnover to produce a null phenotype for the −1/10 mutant in vivo. Because the sub −1/10 mutation produced transcripts that initiated at multiple sites, we deemed it interesting to assess if correct 3′-end formation had been affected similarly. An RNase protection assay was performed on in vitro transcripts using an antisense riboprobe that would recognize SL RNA sequences from nt +38 to nt +196 (Fig. 2 B, schematic). The 3′-end of in vivo synthesized RNAs maps immediately upstream of the T stretch beginning at nt +99. Accordingly, the endogenous SL RNAs present within the nuclear extract protected a fragment of 51 nt (Fig. 2 B). Correctly terminated and/or 3′-end processing in vitrotranscribed SL RNAs would protect a larger, 81-nt fragment because of the presence of the internal 19 nt tag. The 81-nt RNA shown in Fig.2 B demonstrated that the 3′-end of the SL RNA, transcribed from a wt SL RNA gene template, was properly generated in vitro. Interestingly, the 3′-end of the SL RNA transcribed from the sub −1/10 DNA template was also correctly formed. Previousin vivo analysis of SL RNAs by Northern blot analysis is consistent with this finding (7Hartree D. Bellofatto V. Mol. Biochem. Parasitol. 1995; 71: 27-39Crossref PubMed Scopus (34) Google Scholar). The significant reduction of the 81-nt product in the right lane (sub −1/10) compared with the left lane (wt) was consistent with the overall reduction in transcription levels from the mutant template. The 51-nt internal control band (which represents the endogenous RNA) demonstrated that equal amounts of total RNA were included in each RNase protection assay. These results demonstrate the requirement for the −1/10 nt region exclusively in transcription start site selection and not in transcription termination of the SL RNA gene. In the absence of a TATA box to direct start site selection in any of the known, albeit few, characterized trypanosome gene promoters, we tested whether that downstream-most element within the tripartite promoter of the SL RNA gene contributed directly to accurate RNA initiation (20Zawel L. Reinberg D. Annu. Rev. Biochem. 1995; 64: 533-561Crossref PubMed Scopus (389) Google Scholar). A growing collection of higher eukaryotic and yeast genes relies on an Inr element, often without a nearby TATA sequence, for directing proper initiation (21Smale S.T. Biochim. Biophys. Acta. 1997; 1351: 73-88Crossref PubMed Scopus (499) Google Scholar, 22Smale S. Baltimore D. Cell. 1989; 57: 103-113Abstract Full Text PDF PubMed Scopus (1148) Google Scholar, 23Mosch H. Graf R. Braus G. EMBO J. 1992; 11: 4583-4590Crossref PubMed Scopus (14) Google Scholar, 24Furter-Graves E. Furter R. Hall B.D. Mol. Cell. Biol. 1991; 11: 4121-4127Crossref PubMed Scopus (17) Google Scholar, 25Maicas E. Friesen J. Nucleic Acids Res. 1990; 8: 3387-3393Crossref Scopus (25) Google Scholar). As a result of the data shown in Fig. 2 A, lane 2, the downstream-most element maintains functional homology to the metazoan Inr and is now referred to as a t rypanosome Inr, or Inrt. However, a sequence comparison between the metazoan Inr and Inrt shows a distinct difference. In metazoans, a loose consensus exists of YYA(+1)NT/AYY in which it is crucial for the +3 position to be an A or T for optimal Inr activity (26Lo K. Smale S. Gene ( Amst. ). 1996; 182: 13-22Crossref PubMed Scopus (174) Google Scholar). A data base survey of the sequence that flanks SL RNA transcription start sites in related Trypanosomatidae reveals a consensus YYHBYA(+1)ACT in which the C (+3) is invariant. Hence, the A/T (+3) found in metazoans is absent in trypanosomatids. Because of this distinguishing difference between trypanosome and metazoan Inr, it is appropriate to refer the trypanosome Inr as Inrt. To delineate the boundaries of the Inrt element, mutations were introduced in and around the −1/10 nt region (5′-AGACCCTACCA(+1)ACT-3′) of the SL RNA promoter. Initially, each half of the 10 nt region was mutated separately, and mutant templates were used to program nuclear extracts. Fig. 3, lanes 2–4, illustrates the transcription results. Mutation of the −6/10 nt region caused 36% of the transcripts to initiate at the −3 position (lane 4). A comparison of the sequence with the consensus YTHBYA(+1)ACT Inrt showed that a new Inrt had been generated by the substitution of the -6/10 nt region (see Fig. 5). Specifically, the wt sequence from −1/10, which is AGACCCTACCA(+1), had been replaced with TACGTCTACCA(+1). In the mutant construct, a CGTCTA(+1) was recognized at high efficiency to initiate SL RNA transcription. Substitution of the −1/5 nt region (lanes 2 and 3) completely abolished synthesis of properly initiated SL RNAs. Clearly, replacement of the CTACCA(+1) sequence with GATGGA(+1) abolished Inrt function. In the absence of a wt Inrt in the −1/5 mutation, cryptic sites, partially generated by the replacement nucleotides, were recognized by the transcription machinery (see Fig. 5). Consequently, RNAs initiated at several sites, albeit with decreased efficiency. These initiation sites were as follows: CTAACG(+1), located at +8 (relative to the wt (+1) start site); ATGGAA(+1), located at +2; CCGATG(+1), located at −2; AGACCG(+1), located at −5. In the case of the start site utilized with the highest efficiency (the −5 site; 50% of total RNAs produced) the initiating purine (G, in this case), was preceded by an ACC trinucleotide that is identical to that within the wt Inrt. The consistently best utilized start site in the sub −1/10 mutation (the +8 site, 45% of total RNA produced; fastest migrating band inlane 2) also functioned with modest efficiency in the −1/5 mutation (20% of total RNA synthesized; lanes 2 and3). This start site is preceded by a CTAACG(+1) sequence that is identical to the wt CTACCA(+1) region except that the −2 position is an A in place of a C, and the purine that is used as the initiation nucleotide is G in place of A. Thus, conservation of at least three nt within the five nt upstream of the start site is clearly important for Inrt activity. In del-Inrt 2, the nucleotides at positions −6 and −7 nt were altered from CC to GA. This alteration had no effect on start site selection (lane 5), nor did this dinucleotide substitution generate a new Inrt sequence. Taken together, these data show that the Inrt is restricted to the five nt adjacent to the initiating purine. Transcriptional analysis of SL RNA genes in three related trypanosomatids have shown that alterations within the SL RNA sequence had minor effects on both transcription efficiency and start site selection, although these effects were not studied in detail (5Yu M.C. Sturm N. Saito R. Roberts T. Campbell D. Mol. Biochem. Parasitol. 1998; 94: 265-281Crossref PubMed Scopus (27) Google Scholar, 17Günzl A. Ullu E. Dörner M. Fragoso S. Hoffmann K. Milner J. Morita Y. Nguu E. Vanacova S. Wünsch S. Dare A. Kwon H. Tschudi C. Mol. Biochem. Parasitol. 1997; 85: 67-76Crossref PubMed Scopus (71) Google Scholar,27Crenshaw-Williams K. Bellofatto V. Parasitol. Res. 1999; 85: 700-706Crossref PubMed Scopus (5) Google Scholar). As an important component of our Inrt studies, we determined directly if the SL RNA sequence must follow the Inrt immediately. Moreover, by the insertion of four nt, we altered the helical face of the SL RNA sequence relative to the upstream promoter. Any protein-DNA recognition that straddled the upstream and intragenic regions would be disrupted in this mutated template. The insertion mutation, add-C4, did not alter start site selection
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