Gene expression in plastids of higher plants: evolutionary and functional aspects of different RNA polymerases - coordinated assembly of multiprotein-complexes

Plastid gene organisation maintains characteristics typical of its
prokaryotic ancestry. The regulation of plastid gene expression
however strongly deviates from that one its free-living
cyanobacteria relatives. This intriguing complication of plastid
gene expression characteristics is the result of an integration
process of the cellular subgenomes that introduced eukaryotic
traits into the formerly prokaryotic compartment (Herrmann et al.
1997). An interesting example for this process is the transcription
system, which consists of both prokaryotic (PEP) and eukaryotic
(NEP) RNA polymerases. In order to understand, how far the
transcriptional apparatus within plastids was adapted to nuclear
needs, three approaches have been undertaken. Firstly, the tobacco
homologues of the NEP-enzymes known from Arabidopsis were
determined and characterised. Secondly, an extensive transcript
analysis for all plastid operons was carried out with wild-type and
PEP-lacking material in order to assess the contribution of the two
systems to transcription. In order to rapidly screen this plant
material, an array-based technique was established. As no arrays
for the plastid chromosome had been described, the preparation of
the filters, optimisation of hybridisation conditions and probe
preparation together with the use of the proper controls was one of
the main challenges of this work, in particular as macroarrays are
interesting for various other applications. Thirdly, to assess the
regulation of PEP expression, an in vivo analysis of the promoter
for the rpoB operon was performed. According to data from other
groups, this operon is controlled by NEP, which - in addition to
other data - led to the suggestion that PEP is switched on by NEP
(Hajduckiewicz et al., 1997). To test this, point mutations of the
described NEP rpoB promoter (Liere and Maliga; 1999) should be
prepared and introduced into the plastid compartment by particle
transformation. Insight into the regulation of PEP is crucial in
order to understand the interplay of the two RNA polymerase
systems. Finally, in addition to studies on plastid transcription,
later steps in plastid gene expression were examined in the course
of this work as well. As a model to study assembly of a
multisubunit complex, the cytochrome b6/f complex was analysed by a
gene-disruption approach. Deletion and/or insertion mutants of all
plastid encoded subunits of the complex were prepared in order to
evaluate the assembly strategy for the holocomplex.