trans

SL RNA AND U snRNA GENE TRANSCRIPTION IN NEMATODES AND TRYPANOSOMATIDS 61
element, however, had no influence on in vitro
synthesis of correct-sized transcripts.
SL RNA gene transcription in
trypanosomatids
SL RNA genes in trypanosomatids are tandemly
repeated and transcribed monocistronically
by RNA pol II. The SL RNA gene promoter has
been studied in several trypanosomatid species.
The most detailed analyses, which included
transient reporter gene expression and in vitro
transcription assays, were conducted in
Leptomonas seymouri, Leishmania tarentolae
and T. brucei. The trypanosomatid promoters
share no obvious sequence homologies. However,
upstream of the TIS, they contain two distinct
sequence elements instead of the single,
compact element present in the human and
nematode U1 and SL RNA gene promoters
(Figure 3.2). In Lep. seymouri, the upstream
element, termed promoter binding protein 1
element (PBP-1E), is centered at position 65
and the proximal element PBP-2E is located
around position 35. While in L. tarentolae the
corresponding elements are located at the same
positions, the proximal element of T. brucei is
placed 10 bp further upstream. Two observations
suggest that the two promoter elements
have the same function in the three different
trypanosomatid species. Changing the distance
between these elements dramatically
reduced transcription efficiency, and moving
both elements closer or further away from the
TIS impaired correct transcription initiation.
In L. tarentolae, this bipartite promoter suffices
to direct efficient and correct SL RNA gene
transcription. Conversely, mutation of the 10-bp
block just upstream of the TIS both reduced
transcription efficiency and caused aberrant
transcription initiation in Lep. seymouri and
T. brucei. In a more detailed analysis, conducted
in Lep. seymouri, the 6-bp sequence from position
5 to 1 was identified to be responsible
for the observed effects. As specific protein
binding to this sequence motif was detected
by gel shift assays, it was proposed that this
element corresponds to an initiator element.
However, initiator elements have not been
found in U snRNA genes yet. Further experiments
are necessary to determine the exact
role of this element in trypanosomatid SL RNA
genes. In Lep. seymouri, specific DNA–protein
complexes have also been identified at promoter
elements PBP-1E and PBP-2E. Gel-shift
assays and DNase I footprinting demonstrated
that PBP-1E specifically and efficiently binds
the putative transcription factor PBP-1. Purification
of PBP-1 revealed three polypeptides
of 57 kDa, 46 kDa, and 36 kDa, reminiscent of
the multi-subunit SNAPc in higher eukaryotes,
and photocrosslinking suggested that the
46 kDa polypeptide binds to the promoter
domain. Furthermore, it was shown that binding
of PBP-1 to the SL RNA gene promoter and
the integrity of the second promoter element
PBP-1E are prerequisites for the formation of a
larger and more stable complex, suggesting
that the two upstream promoter elements
cooperate in the formation of a stable transcription
initiation complex. Accordingly, the
integrity of both elements was essential to
stably bind and sequester a trans-activating
factor in transcription competition experiments
conducted in T. brucei. In L. tarentolae,
however, gel-shift assays have thus far identified
specific protein binding only to PBP-1E and
not to the second promoter element.
A conserved feature of trypanosomatid
SL RNA genes is a T-run a few basepairs
downstream of the SL RNA coding region. In
L. tarentolae, the T-run was identified as the
transcription termination determinant. Hence,
RNA pol II-mediated SL RNA gene transcription
in trypanosomatids appears to terminate
MOLECULAR BIOLOGY