trans

CLASS I TRANSCRIPTION IN TRYPANOSOMATIDS 53
The rDNA promoter of T. brucei, although
less well characterized, has a similar organization
(Figure 3.1). The essential region ranges
from position 257 to 13 and most likely contains
four domains like the procyclin ES promoter.
While domain I (42/13) and domain
II (62/53) were unambiguously identified
by a block substitution analysis in the proximal
promoter region, 5 promoter deletions indicated
the presence of domains III and IV in the
distal part of the promoter. Subsequently,
domain IV was characterized in more detail by
in vitro transcription competition experiments,
which uncovered a surprising piece of information:
a linear DNA fragment containing the
rDNA promoter competed transcription of the
SL RNA gene, which served as a control template
in the competition assays. This unexpected
finding suggested that rDNA and SL
RNA gene promoters, which recruit different
RNA pols, bind a common trans-activating
transcription factor. A closer look at the rDNA
promoter revealed two sequence blocks in the
most distal promoter region which, in opposite
orientation, are homologous to two essential
SL RNA gene promoter elements (Figure 3.1;
and see below). When these sequences were
mutated, the rDNA fragment was unable to
compete SL RNA gene transcription in vitro
and the rDNA promoter lost its ability to direct
efficient gene expression in vivo, demonstrating
the functional importance of these elements
in the rDNA promoter. Domain IV is
dispensable for efficient transcription in vitro
indicating that it is functionally analogous to
domain IV of the procyclin ES promoter. However,
domains IV of rDNA and procyclin ES promoters
are distinct elements because they share
no sequence homology, and the procyclin ES
promoter does not bind the activator of SL RNA
gene transcription. Hence, domain IV of the procyclin
ES promoter presumably interacts with a
specific protein.
In addition to their study in T. brucei, rDNA
promoters have been investigated by transient
transfection assays in several Leishmania
species. The most detailed study was conducted
in L. donovani, and revealed a structure surprisingly
different from the corresponding T. brucei
promoter. In L. donovani, the 69-bp region
upstream of the TIS is sufficient to direct full
transcriptional activity, and it contains two distinct
promoter domains (Figure 3.1). Interestingly,
transcription efficiency was reduced when
the sequence around the initiation site was
changed. However, since mutation of the initiation
nucleotide itself may have a dramatic
effect on transcription efficiency, it is currently
not clear whether the L. donovani rDNA
promoter includes an element at this site essential
for transcription complex assembly.
VSG ES promoters represent the third type
of class I promoters in T. brucei. Several promoter
sequences have been determined, and
the few single nucleotide polymorphisms
found appear to have no influence on transcription
activity. Interestingly, the structure
of the VSG ES promoter closely resembles
that of the L. donovani rDNA promoter; it is
of the same short size and it has the same
two-domain structure (Figure 3.1). Hence, in
T. brucei, the VSG ES promoter differs significantly
from procyclin ES and rDNA promoters
because it lacks domains III and IV. Moreover,
transcription competition experiments
in vitro showed that VSG ES promoter domains
I and II, in contrast to their counterparts in the
procyclin ES promoter, are essential for and
cooperate in stable binding of transcription
factors. Hence, it appears that domains I and
II of these two promoters are not functionally
analogous.
To date, it has not been clarified whether
promoters of metacyclic VSG ES represent a
fourth type of class I gene promoters in T. brucei.
This may be the case because mVSG gene
MOLECULAR BIOLOGY