A novel intermediate in transcription initiation by human mitochondrial RNA polymerase
Yaroslav I. Morozov
1
2
Karen Agaronyan
1
2
Alan C. M. Cheung
0
1
Michael Anikin
1
2
Patrick Cramer
0
1
Dmitry Temiakov
1
2
0
Gene Center and Department of Biochemistry, Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universita t M unchen
, Feodor-Lynen-Strasse 25,
81377 Munich, Germany
1
Present address: Alan C. M. Cheung,
Institute of Structural and Molecular Biology, University College London/Birkbeck
, Malet Street, London WC1E 7HX,
UK
2
Department of Cell Biology, School of Osteopathic Medicine, Rowan University, Medical Center Dr
, Stratford,
NJ 08084, USA
The mitochondrial genome is transcribed by a single-subunit T7 phage-like RNA polymerase (mtRNAP), structurally unrelated to cellular RNAPs. In higher eukaryotes, mtRNAP requires two transcription factors for efficient initiationTFAM, a major nucleoid protein, and TFB2M, a transient component of mtRNAP catalytic site. The mechanisms behind assembly of the mitochondrial transcription machinery and its regulation are poorly understood. We isolated and identified a previously unknown human mitochondrial transcription intermediatea pre-initiation complex that includes mtRNAP, TFAM and promoter DNA. Using proteinprotein cross-linking, we demonstrate that human TFAM binds to the N-terminal domain of mtRNAP, which results in bending of the promoter DNA around mtRNAP. The subsequent recruitment of TFB2M induces promoter melting and formation of an open initiation complex. Our data indicate that the pre-initiation complex is likely to be an important target for transcription regulation and provide basis for further structural, biochemical and biophysical studies of mitochondrial transcription.
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INTRODUCTION
Mitochondrial transcription does not fit the paradigm of
eukaryotic or prokaryotic transcription systems, as
it relies on a single subunit T7 phage-like mtRNAP.
However, unlike the T7 system, transcription initiation
by mtRNAP involves a number of additional factors,
suggesting a more complex organization that likely reflects
regulatory needs by the mitochondrial system (1,2).
Cellular multi-subunit polymerases form an array of
transient complexes along the pathway to transcription
initiation (37). These intermediates serve as important
targets for regulation by presenting a specific
conformation of RNAP to various regulatory factors. Binding of
these factors at early stages of transcription is an
important mechanism that affects cellular physiology and
development, and this phenomenon has been well studied in a
number of systems (8,9). As noted above, mtRNAP also
requires auxiliary factors for transcription initiation;
however, the mechanisms of promoter recognition,
binding and melting by the mtRNAP must be distinct
from those established for phage T7 RNAP (1012),
which does not require such factors, and in which
formation of stable transcription intermediates has not been
reported (13,14).
While most eukaryotic organisms contain mitochondria,
the basal mitochondrial transcription machinery appears
to have evolved differently in lower and higher eukaryotes.
Thus, the yeast core transcription system is composed of
mtRNAP and a single transcription initiation factor,
Mtf1, which is implicated in promoter melting (15,16).
In contrast, the mammalian core transcription apparatus
contains, in addition to mtRNAP and TFB2M (a
functional analog of Mtf1), an abundant mitochondrial
protein, TFAM (17,18) that is a major component of the
mitochondrial nucleoid and is required for mtDNA
organization and maintenance; knockout of the latter
protein results in a dramatic loss of mtDNA and
disruption of oxidative phosphorylation (19,20). While yeast
mitochondria also contain TFAM, it has no apparent
role in transcription and serves only as a nucleoid
protein, likely due to truncation (as compared with
human TFAM) of a C-terminal tail domain that has
been implicated in transcription activation in human
mitochondria (21). Human TFB2M is transiently
associated with mtRNAP during initiation and interacts
with the templating DNA base and the priming substrate
(22). Both TFB2M and Mtf1 have been implicated in
regulation of transcription initiation in response to
variations in cellular ATP concentrations (22,23).
Despite recent progress in structural studies of human
mtRNAP and TFAMDNA complexes (2,2426), the
mechanisms of assembly of the mitochondrial
transcription initiation complex are poorly understood and are
somewhat controversial. It has been suggested that
TFAM, which leaves a clear footprint on two major
human mitochondrial promoters, termed LSP and HSP1
(21,27), interacts via its C-terminus with TFB2M,
implicating the latter in recruiting mtRNAP to its
promoter (28). On the other hand, it has been postulated
that the mitochondrial core transcription system includes
only mtRNAP and TFB2M, and that TFAM is
dispensable for the initiation process and acts to stimulate basal
levels of transcription from both the LSP and HSP1
promoters (29) (...truncated)