trans

CLASS I TRANSCRIPTION IN TRYPANOSOMATIDS 55
reported in which such proteins activate or
repress transcription. In T. brucei, however, the
functional significance of single-stranded DNA–
protein complexes for transcription remains
to be determined. Basal transcription factors
of other eukaryotes interact with doublestranded
DNA. T. brucei may be no exception
here because specific, double-stranded DNA–
protein complexes were identified with probes
spanning the procyclin ES promoter domain I
or both domains of the VSG ES promoter.
Transcription termination
The mechanism of RNA pol I transcription
termination is conserved between yeast and
mammals, and requires a terminator consisting
of a termination factor and its specific
DNA binding site. Termination is caused by
specific interactions of RNA pol I and the
termination factor and depends on the right
orientation of the terminator. Berberof and
colleagues used transient transfection assays
to search for termination determinants in a
procyclin ES. By cloning putative terminatorcontaining
sequences between the procyclin
ES promoter and a reporter gene, they were
able to identify three regions which efficiently
reduced reporter gene expression, presumably
by stopping RNA pol I. In accordance
with the general model, these DNA fragments
were functional in an orientation-dependent
manner and did not inhibit RNA pol II-driven
gene expression. Interestingly, in one of these
fragments, an important sequence motif was
identified which resembled domain II of the
VSG ES promoter and which interacted with
the same 40 kDa single-stranded DNA binding
factor (see above, Promoter structures). While
further experiments are necessary to assess
the role of this single-stranded DNA–protein
complex in transcription termination, the
connection between terminator and promoter
elements made in this study is typical for
rDNA repeat units of yeast and higher eukaryotes.
These gene units contain termination
signals downstream of the ribosomal genes, as
expected, but additionally possess a single terminator,
denoted as proximal terminator or
T 0 , approximately 200 bp upstream of the TIS.
The proximal terminator can be multifunctional,
but in all cases analyzed it is involved in
remodeling the chromatin structure of the promoter
region. In yeast, the termination signal
and proximal terminator is the Reb1p binding
domain (Figure 3.1). Since domains IV of the
T. brucei procyclin ES and rDNA promoters
may be functionally analogous to the proximal
terminator (see section on Promoter structures),
they may also function in transcription
termination.
RNA pol I transcription machinery
The conventional way to characterize a transcription
component involves purification of
the protein, polypeptide sequencing, and cloning
of the respective gene. However, the very low
abundance of most transcription factors makes
them difficult to purify and, in trypanosomatids,
no polypeptide involved in RNA pol I-mediated
transcription has yet been discovered in this
way, despite the characterization of specific
band-shifts and the development of an in vitro
transcription system.
Another strategy is data mining of genome
databases for conserved sequence motifs to
identify homologs of polypeptides already
characterized in other eukaryotes. Thus far,
this has been successful for bona fide RNA pol
I subunits. Many eukaryotic RNA pol subunits
are functionally conserved and homologs
exist even in archaebacteria. By exploiting this
high degree of conservation, the gene of the
MOLECULAR BIOLOGY