trans

56 TRANSCRIPTION
largest subunit of T. brucei RNA pol I was identified
and characterized. It is a single-copy gene
and encodes a 1781 amino acid-long polypeptide
with a predicted molecular weight
of 197 kDa. The deduced amino acid sequence
harbors RNA pol I-specific regions as well as
eight conserved domains present in all eukaryotic
RNA pol largest subunits. Recently, we
used a partial T. brucei sequence similar to the
RNA pol I second largest subunit (gene bank
accession no. W84090) to clone the complete
cDNA of this polypeptide. The deduced amino
acid sequence contains conserved domains of
second largest subunits as well as sequences
homologous to RNA pol I signatures defined
in higher eukaryotes. The predicted molecular
weight of 180 kDa of this polypeptide, which
was confirmed by partial purification of RNA
pol I, is unusually large due to an approximately
300 amino acid-long N-terminal domain
which is not present in homologs of other
eukaryotes. If this extra domain is of functional
significance, it may serve as a target
against the parasite.
While the script-based functional classification
of sequences present in trypanosomatid
genome databases identified several putative
RNA pol subunit genes (see the Parasite Proteome
Analysis web page at http://www.ebi.
ac.uk/parasites/proteomes.html), no similarities
to auxiliary factor genes have yet been
detected. This may not be possible at all
because in contrast to most RNA pol subunits,
class I factors are not well conserved among
eukaryotes. Exceptions to this rule are TBP
which generally is part of the class I basal
machinery and histones H3 and H4 which are
subunits of the yeast upstream activation
factor. In trypansomatids, a TBP homolog has
not been identified thus far, and it remains
to be determined whether histones play a role
as transcription factor components in these
organisms.
CLASS II TRANSCRIPTION OF
PROTEIN CODING GENES
Trichomonas vaginalis and
Giardia lamblia
T. vaginalis and G. lamblia belong to phylogenetic
lineages which diverged very early from
the main eukaryotic lineage. In T. vaginalis,
the sequence surrounding the TIS of protein
coding genes is conserved and transient
reporter gene expression assays have shown
that it is an essential promoter element
directing accurate transcription. In addition,
the sequence specifically binds a nuclear factor
and, therefore, was identified as an initiator
element. Initiator elements have been
characterized predominantly in mammalian
systems. They interact with the basal transcription
machinery and are important for
transcription initiation especially in TATA-less
promoters. Interestingly, a mammalian initiator
element could functionally substitute for
the Trichomonas element, suggesting that
initiator elements are highly conserved and
evolved very early in eukaryotic evolution. In
G. lamblia, the TIS of protein coding genes is
surrounded by an A/T-rich sequence which, as
has been shown for several genes, is essential
for in vivo expression of a reporter gene and,
by itself, is able to direct a low level of expression.
In contrast to the T. vaginalis initiator
element, the A/T-rich sequence is not well
conserved and cannot be substituted by a
mammalian initiator element. In both G. lamblia
and T. vaginalis genes, promoter elements
were identified in the region upstream of the
TIS. In G. lamblia, a consensus TATA element
has not been identified, but A/T-rich elements,
located between positions 20 and
60, were shown to be important transcription
determinants. T. vaginalis promoters
apparently lack TATA boxes. Analysis of the
MOLECULAR BIOLOGY