Molecular basis of RNA polymerase III transcription repression by Maf1 & Structure of human mitochondrial RNA polymerase

Topic I Molecular basis of RNA polymerase III transcription
repression by Maf1 RNA polymerase III (RNAP III) is a conserved
17-subunit enzyme that transcribes genes encoding short
untranslated RNAs such as transfer RNAs (tRNAs) and 5S ribosomal
RNA (rRNA). These genes are essential and involved in fundamental
processes like protein biogenesis; hence RNAP III activity needs to
be tightly regulated. RNAP III is repressed upon stress and this is
regulated by Maf1, a protein conserved from yeast to humans. Many
stress pathways were shown to converge on Maf1 and result in its
phosphorylation, followed by its nuclear import and eventual
repression of RNAP III activity. However, the molecular mechanisms
of this repression activity were not known at the beginning of
these studies. This work establishes the mechanism of RNAP III
specific transcription repression by Maf1. The crystal structure of
Maf1 was solved. It has a globular fold with surface accessible NLS
sequences, which sheds new light on already published results and
explains how stress-induced phopshorylation leads to import of Maf1
into the nucleus. Additionally, cryo EM studies and competition
assays show that Maf1 binds RNAP III at its clamp domain and
thereby induces structural rearrangements of RNAP III, which
inhibits the interaction with Brf1, a subunit of the transcription
initiation factor TFIIIB. This specifically impairs recruitment of
RNAP III to its promoters and implies that Maf1 is a repressor of
transcription initiation. Competition and transcription assays show
that Maf1 also binds RNAP III that is engaged in transcription,
leaving RNAP III activity intact but preventing re-initiation.
Topic II Structure of human mitochondrial RNA polymerase The
nuclear-encoded human mitochondrial RNAP (mitoRNAP) transcribes the
mitochondrial genome, which encodes rRNA, tRNAs and mRNAs. MitoRNAP
is a single subunit (ss) polymerase, related to T7 bacteriophage
and chloroplast polymerases. All share a conserved C-terminal core,
whereas the N-terminal parts of mitoRNAP do not show any homology
to other ss RNAPs. Unlike phage RNAPs, which are self-sufficient,
human mitoRNAP needs two essential transcription factors for
initiation, TFAM and TFB2M. Both of these factors are likely to
control the major steps of transcription initiation, promoter
binding and melting. Thus human mitoRNAP has evolved a different
mechanism for transcription initiation and exhibits a unique
transcription system. Structural studies thus far concentrated on
the nuclear enzymes or phage RNAPs, whereas the structure of
mitochondrial RNA polymerase remained unknown. The structural
organization of human mitoRNAP and the molecular mechanisms of
promoter recognition, binding and melting were subject of interest
in these studies. In this work the crystal structure of human
mitoRNAP was solved at 2.4 Å resolution and reveals a T7-like
C-terminal catalytic domain, a N-terminal domain that remotely
resembles the T7 promoter-binding domain (PBD), a novel
pentatricopeptide repeat (PPR) domain, and a flexible N-terminal
extension. MitoRNAP specific adaptions in the N-terminus include
the sequestering of one of the key promoter binding elements in T7
RNAP, the AT-rich recognition loop, by the PPR domain. This
sequestration and repositioning of the N-terminal domain explain
the need for the additional initiation factor TFAM. The highly
conserved active site within the C-terminal core was observed to
bind a sulphate ion, a well known phosphate mimic, and thereby
suggests conserved substrate binding and selection mechanisms
between ss RNAPs. However, conformational changes of the active
site were observed due to a movement of the adjacent fingers
subdomain. The structure reveals a clenching of the active site by
a repositioned fingers subdomain and an alternative position of the
intercalating -hairpin. This explains why the conserved
transcription factor TFB2M is required for promoter melting and
initiation. A model of the mitochondrial initiation complex was
build to further explore the initiation mechanism, and to
rationalize the available biochemical and genetic data. The
structure of mitoRNAP shows how this enzyme uses mechanisms for
transcription initiation that differ from those used by phage and
cellular RNAPs, and which may have enabled regulation of
mitochondrial gene transcription and adaptation of mitochondrial
function to changes in the environment.