trans

54 TRANSCRIPTION
expression is regulated exclusively at the level
of transcription and is different from that
of bloodstream-form VSG genes. In addition,
mVSG ES promoter sequences are less well
conserved and deviate significantly from the
consensus sequence of bloodstream-form VSG
ES promoters. On the other hand, mVSG ES
promoters are functional when truncated to
the same short size as their counterparts in
bloodstream-form VSG ESs and they appear to
have the same two-domain structure. However,
analysis of the MVAT7 mVSG ES promoter
suggested that the promoter domains are not
located at the same positions as their counterparts
in bloodstream forms. In summary, class I
promoters of trypanosomatids can be divided
into two groups: the T. brucei rDNA and procyclin
ES promoters represent the first group
which is characterized by a multi-domain
structure resembling that of the yeast rDNA
promoter. The second group comprises the
L. donovani rDNA and T. brucei VSG ES promoters
which are very short, not extending further
upstream than position 70, and possess
two distinct domains upstream of the TIS.
A two-domain structure is reminiscent of vertebrate
rDNA promoters, but in these organisms
domain II is much larger and located
approximately 90 bp further upstream. Therefore,
the structure of the two trypanosomatid
promoters in a strict sense does not resemble
any known class I promoter.
Finally, as has been shown in other eukaryotic
organisms, rDNA transcription is stimulated
by enhancer elements which work
relatively independently of distance and which
are typically arranged as tandem repeats. In this
respect, it is interesting to note that repetitive
elements are present in trypanosomatid rRNA
gene units. In several Leishmania species and
in Trypanosoma cruzi, tandem repeats were
identified close to the promoter region, and in
the T. brucei rRNA gene unit, repetitive elements
are located approximately 9 kb upstream of
the promoter region. Furthermore, VSG ESs in
T. brucei have a 50-bp repeat array approximately
1.4 kb upstream of the TIS whereas no
repeats have yet been reported for mVSG and
procyclin ESs. Interestingly, in L. chagasi, the
repeats moderately enhanced transient rDNA
promoter-directed expression of a reporter
gene, whereas those of the L. donovani rDNA
promoter and of the T. brucei VSG ES promoter
exhibited no enhancer effect. It should be
noted though that, in the yeast rDNA promoter,
the enhancer repeats were only functional in a
chromosomal context and not in an episomal
vector construct. Hence, it still needs to be
clarified whether repetitive elements in trypanosomatid
class I gene units function as
transcriptional enhancers.
DNA–protein interactions at
promoter domains
In a first step to characterize the transcription
machinery in T. brucei, the interaction of proteins
with class I gene promoter elements has
been investigated by gel retardation assays,
and specific band-shifts were reported for all
three promoter types. Interestingly, for both
procyclin and VSG ES promoters, specific
protein binding to single-stranded promoter
sequences was observed. A polypeptide of
40 kDa was identified binding to the antisense
and sense strands of VSG ES promoter domains
I and II, respectively, due to an inverted
sequence motif present in these domains.
Furthermore, competition in DNA/protein
complex formation suggested that the same
protein binds to the antisense strand of domain
I in the rDNA and procyclin ES promoters. The
involvement of single-stranded DNA-binding
proteins in transcription regulation is not
unprecedented, and several cases have been
MOLECULAR BIOLOGY