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 m 7 G 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 doublestranded 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.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. 1). The protein-coding portion is transcribed as a single primary mRNA, often containing several open reading frames flanked by 5Ј-and 3Ј-untranslated regions. The capped 5Ј-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 5Ј-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 (2). Addition of the SL provides each mRNA with an m 7 G cap as well as four extensively methylated nucleotides, at positions 1-4 within the 39-nt SL RNA (3).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. ␣-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 5Ј-and 3Ј-ends, indicating that both transcription initiation and termination regulate the accumulation of SL RNA. SL RNA expression has been monitored using independe...

Transcription Initiation and Termination on Leishmania major Chromosome 3

“…The 247-bp region which separates the most 5Ј TSSs for gene 1 and gene 2 does not show any substantial sequence identity with the region upstream of the TSSs of gene 98, the putative promoter region for LmjF chr1 (19), or the SL promoter region of chr2 (16). As previously seen for chr1, no TATA box or any other typical Pol II promoter elements are apparent, and the GC content (55%) is similar to that of the entire strand-switch region (51% GC), although it is somewhat lower than chr3 (and the genome) as a whole (63%).…”

Spliced Leader RNA Gene Transcription in Trypanosoma brucei Requires Transcription Factor TFIIH

“…The detailed characterization of the SL RNA gene promoter in three trypanosomatid species revealed a bipartite upstream sequence element (USE) (8,14,38) and an initiator (21). A focus of trypanosomatid SL RNA biology that has recently emerged is the characterization of the transcription machinery required for SL RNA synthesis.…”

Characterization of a Multisubunit Transcription Factor Complex Essential for Spliced-Leader RNA Gene Transcription in Trypanosoma brucei

“…To accommodate the high synthesis rate, a trypanosome cell harbors approximately 200 SL RNA gene copies, which are transcribed by RNA polymerase II in a monocistronic fashion (3). The anatomy of the SL RNA gene promoter has been meticulously investigated in the three trypanosomatid species Trypanosoma brucei (7), Leptomonas seymouri (8,13), and Leishmania tarentolae (38); it is conserved and consists of a bipartite upstream sequence element (USE) and a putative initiator element (23) at the transcription initiation site.…”