Bulged residues promote the progression of a loop–loop interaction to a stable and inhibitory antisense–target RNA complex
Fabrice A. Kolb
1
Eric Westhof
1
Chantal Ehresmann
1
Bernard Ehresmann
1
E. Gerhart H. Wagner
0
1
Pascale Romby
1
S-
1
Uppsala
1
Sweden
1
0
Institute of Cell and Molecular Biology, Biomedical Center, Uppsala University
, Box 596, Husargatan 3
1
UPR 9002 du CNRS,
Institut de Biologie Molculaire et Cellulaire
, 15 rue Rene Descartes, 67084 Strasbourg Cedex
In several groups of bacterial plasmids, antisense RNAs regulate copy number through inhibition of replication initiator protein synthesis. These RNAs are characterized by a long hairpin structure interrupted by several unpaired residues or bulged loops. In plasmid R1, the inhibitory complex between the antisense RNA (CopA) and its target mRNA (CopT) is characterized by a four-way junction structure and a side-by-side helical alignment. This topology facilitates the formation of a stabilizer intermolecular helix between distal regions of both RNAs, essential for in vivo control. The bulged residues in CopA/CopT were shown to be required for high in vitro binding rate and in vivo activity. This study addresses the question of why removal of bulged nucleotides blocks stable complex formation. Structure mapping, modification interference, and molecular modeling of bulged-less mutant CopA-CopT complexes suggests that, subsequent to loop-loop contact, helix propagation is prevented. Instead, a fully base paired loop-loop interaction is formed, inducing a continuous stacking of three helices. Consequently, the stabilizer helix cannot be formed, and stable complex formation is blocked. In contrast to the fourway junction topology, the loop-loop interaction alone failed to prevent ribosome binding at its loading site and, thus, inhibition of RepA translation was alleviated.
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Many antisense RNAs are regulators of plasmid copy number
(1). Plasmid R1 belongs to the IncFII family of plasmids which
shares the overall genetic organization with respect to
replication control functions. In plasmid R1, replication is negatively
controlled at the translational level by binding of the antisense
RNA (CopA) to its target site (CopT) in the leader region of the
repA mRNA, 80 nt upstream of the repA start codon (2).
Synthesis of the replication initiator protein RepA requires
translation of a short leader peptide (tap), encoded upstream of
repA. Binding of CopA prevents tap translation by occluding
ribosome binding at the tap ribosome binding site (RBS), and
thereby repA expression is inhibited (35). Interestingly,
several copy number control antisense RNAs which all inhibit
translation of replication initiator proteins have structural
properties similar to those of CopA. These RNAs are fully
complementary to their target site and contain a long stemloop
structure. For proper control, the intracellular concentration of
the antisense RNA must be a measure of the plasmid
concentration: this is ensured by constitutive synthesis and a short
half-life of these RNAs (6). Furthermore, efficiency of in vivo
control is correlated with high rates of antisense RNA binding
to its target site [in the order of 106 M1s1 (7)]. This class of
antisense RNAs does not require trans-acting proteins to
promote inhibition. Thus it can be speculated that the structure
of these antisense RNAs has evolved to be optimized for the
critical steps that determine their regulatory functions.
Kinetic studies have shown that the initial step of antisense
RNA binding involves a looploop interaction mediated by a
limited number of WatsonCrick base pairs, the so-called
kissing complex [R1 (8); pMU720 (9); ColIb-P9 (10,11)]. In
plasmid R1, we have recently shown that this looploop
interaction is rapidly converted into a stable and inhibitory complex
formed through a hierarchy of distinguishable intermediates,
and that the formation of a full duplex in vitro is too slow to be
of biological relevance (1214). The major product of the
binding reaction adopts an unusual structure characterized by a
four-helix junction. Its formation involves extensive breakage
of intramolecular base pairs to promote the formation of two
intermolecular helices (see Fig. 1) (13). This conversion is
essential for the formation of an irreversible complex and for
the activity of the antisense RNA in vivo (14). We proposed
previously that the four-way junction structure could promote
a side-by-side helical alignment to allow formation of a third
long intermolecular helix, involving the 5 tail of CopA and the
complementary region of CopT (13). Recent data indicated
that a topology, strikingly similar to that of the stable CopA
CopT complex, may be present in complexes of all R1-related
plasmids (11,15). This structure completely and irreversibly
inhibits repA translation, mainly by occluding ribosome
binding [e.g. pMU720 (16); R1 (5); Col1b-P9 (17)].
In addition to the recognition loops and the single-stranded
regions required for stable complex formation, these antisense
and target RNAs carry internal loops or unpaired residues in
the m (...truncated)