We have used nuclear run-on, RT-PCR, and transient-transfection analyses to characterize transcription initiation and termination of LmjF chr3. Our data suggest that, like chr1 (19
), specific Pol II transcription starts upstream of the most-5′ gene of the two long polycistronic clusters. We have also identified a region where Pol III transcription starts for a tRNA gene located at the convergence of these two gene clusters. Termination of Pol II transcription on both DNA strands, as well as Pol III transcription of the tRNA, seems to occur within this region. Thus, we have now characterized the transcriptional organization of two entire chromosomes and have identified sequences involved in both Pol II and Pol III transcription initiation and termination.
Identification and characterization of the Pol II promoters that drive the expression of protein-coding genes in trypanosomatids has proven to be an elusive goal, complicated by factors such as relatively low transcriptional activity and rapid processing of primary transcripts. Putative promoters for actin
), and GARP
) genes have been reported in Trypanosoma brucei
and Trypanosoma congolense
, although these conclusions remain controversial (5
). Until recently, the only Pol II promoter region that has been extensively characterized is the one driving the expression of the SL RNA (10
). These findings, along with the apparent lack of regulation of Pol II transcription and the observation that episomal molecules are transcribed on both strands in Leishmania
), have led to the hypothesis that Pol II has low specificity in trypanosomatids and that transcription of protein-coding genes can start indiscriminately throughout the genome (14
). However, transcriptional analysis of the entire LmjF chr1 (19
) showed that the coding strand-specific Pol II transcription that initiated in the strand-switch region between the two long polycistronic gene clusters was at least 10-fold higher than any nonspecific transcription which may have initiated randomly throughout the chromosome. These studies localized several transcription initiation sites within a <100-bp region, and transfection studies support the presence of a bidirectional promoter in this region.
The results reported here for similar analyses of LmjF chr3 show that transcription of the coding strand was ~8-fold higher than that of the noncoding region and that specific Pol II transcription initiates in the strand-switch region between LmjF3.0010 (gene 1) and LmjF3.0020 (gene 2), near the left telomere, and upstream of LmjF3.0980 (gene 98) at the right telomere (Fig. and ). In addition, there may be another Pol II TSS in the large intergenic region upstream of LmjF3.0680 (gene 68). Since we did not include fragments from all the genes on chr3, we cannot formally exclude the possibility that there are other TSSs in other areas of the chromosome. As for chr1, several TSSs were mapped in the strand-switch region between LmjF3.0010 and LmjF3.0020, the most 5′ of which were separated by only 247 bp (Fig. ). The region of initiation of transcription at the right telomere appears to be unidirectional (away from the telomere), but it also has multiple TSSs. There is no substantial sequence homology between the regions of transcription initiation identified on LmjF chr1 and chr3, and they do not contain any typical Pol II promoter elements. Although each region contains one or two G or C tracts, which can also found in the SL promoter region of chr2, similar-sized G or C tracts are also found in other intergenic regions throughout the genome. Thus, rigorously conserved sequence recognition sites do not appear to be required for Pol II transcription initiation in Leishmania.
Nuclear run-on and transient-transfection experiments (Fig. and ) indicated the presence of a Pol III promoter for the tRNA gene in the strand-switch region between the two large convergent gene clusters on chr3, although the drug inhibition studies were not totally conclusive (Fig. ). Assays performed in the presence of 80 μM tagetitoxin, a concentration reported to inhibit Pol III in other trypanosomatids (8
), did not have any appreciable effect on the hybridization signal (data not shown) and the inhibition obtained with 160 μM tagetitoxin was only 52%. Others have also reported conflicting results when trying to identify the polymerase involved in transcription of other small RNA genes in trypanosomatids (12
). The results obtained here could be explained by the fact the 339-nt tRNA-gene fragment contains 145 nt upstream of the 73-nt tRNA gene and thus this region is most likely transcribed by Pol II (Fig. ). Hence, the top strand of the tRNA-gene region of chr3 appears to be transcribed by both Pol II and Pol III, while the bottom strand is transcribed only by Pol II. The noncoding (bottom) strand of the tRNA-gene cluster from chr23 is also transcribed by Pol II, but it appears that the coding strand may be transcribed by Pol III only (Fig. ).
Comparison with tRNA sequences from other trypanosomatids and Saccharomyces cerevisiae
indicated that the LmjF3.tRNALys.01
gene contains the two DNA elements, box A and box B (Fig. ), typically located downstream of TSS in tRNA-gene promoters in eukaryotes (9
). A few other Pol III promoters have been characterized in trypanosomatids: in T. brucei
the genes coding for the small nuclear RNAs (snRNAs) U2, U3, and U6, as well as the 7SL RNA, are transcribed by Pol III (8
). All these genes have a divergently oriented tRNA gene in their 5′-flanking region, and boxes A and B from the neighbor tRNA genes are essential for expression of the snRNAs and the 7SL RNA (24
). The 7SL RNA gene from Leptomonas collosoma
also requires a companion tRNA gene for efficient expression (3
). In most cases, the snRNAs and 7SL RNA genes also require intragenic regulatory elements to achieve a good level of expression (3
). We have not yet ruled out the possibility that there is an snRNA gene adjacent to the LmjF3.tRNALys.01
gene, although none is apparent from the sequence.
In higher eukaryotes, mRNA (Pol II) transcription termination is a complex process that depends on the presence of a functional poly(A) signal as well as other downstream signals and factors (30
). The polyadenylation sites for LmjF3.0680
(gene 68) and LmjF3.0690
(gene 69) were mapped by RT-PCR. Three polyadenylation sites were localized 145, 155, and 336 bp downstream of the gene 68 stop codon, and two sites were mapped 368 and 440 bp downstream of the gene 69 stop codon (Fig. ). Nuclear run-on analysis indicated that Pol II-mediated transcription of both large polycistronic gene clusters appears to terminate in the strand-switch region between these convergent clusters in the same vicinity as the Pol III-mediated initiation and termination for the tRNA gene. This conclusion was supported by RT-PCR experiments which identified a region within the tRNA gene where Pol II transcription terminates on the top strand. Interestingly, all the termination sites are located in the base-paired region of the anticodon arm of the tRNA. Transient-transfection experiments (Fig. ) and RNase protection assays (data not shown) also support the role of the tRNA-gene region in transcription termination. In most tRNA genes, simple clusters of four or more T residues act as signals to terminate transcription (28
). Our data indicate that the run of four Ts located immediately downstream of the LmjF3.tRNALys.01
gene is involved in Pol III transcription termination (Fig. ). The T tract might also be involved in Pol II termination, in conjunction with other sequences such as the tRNA anticodon arm region. We have not yet determined the exact location of transcription termination on the bottom strand, although the nuclear run-on and RNase protection data suggest that it occurs within the tRNA-gene region. In Leishmania tarentolae
, it has been shown that transcription of the SL-RNA gene (transcribed by Pol II) is terminated by the T tract located downstream of the gene (38
). It is interesting that the region containing the LmjF3.tRNALys.01
gene was also able to terminate Pol I transcription in transient-transfection studies. Although Pol I does not transcribe chr3, the fact that the tRNA-gene region has the capacity to partially terminate (or attenuate) Pol I transcription supports the role of this region in termination of transcription. It is possible that the tRNA-gene region contains sequences that potentially promote the release of any RNA polymerase from the template DNA. Similar studies in T. brucei
found that a 2-kb region in the PARP A
transcription unit, was able to terminate Pol I transcription (from rRNA
, or VSG
promoters), but was unable to block transcription by Pol II (4
In Saccharomyces cerevisiae
, improper termination of two convergent genes resulted in the reduced expression of both genes by a mechanism known as transcriptional collision (29
). The presence of a termination region between the two convergent polycistronic units on chr3 from LmjF suggests that Leishmania
, like S. cerevisiae
, might require separation of adjacent Pol II transcription units by proper termination signals to avoid transcriptional collision. It has been previously speculated that Pol III-gene regions might act as Pol II terminators in T. brucei
, since Pol III genes have been found at the 3′ boundary of Pol II transcription units in several cases (17
). This also appears to be the case in many (but not all) of the convergent strand-switch regions in LmjF (unpublished data).
The findings reported in this work significantly advance our understanding of transcription of protein-coding genes in trypanosomatids. Our data suggest that specific transcription generally starts in the strand-switch region between the first genes in two “divergent” gene clusters and terminates in the strand-switch region where the polycistronic clusters converge. The latter is often associated with Pol III transcription initiation and termination of tRNAs and/or other small RNAs. Since the trypanosomatid genome sequencing projects have revealed that most genes are organized into large clusters (22
), the number of regions where Pol II transcription initiates in these organisms is low compared to other eukaryotes—probably only a few per chromosome.