SUPPLEMENTARY INFORMATION
Additional commentary on features of the structure
As described in the main text, the N-terminal half of E. coli RNase E
encompasses the catalytic function of the enzyme1-3, and this portion resembles
closely the paralogue, RNase G4,5. The C-terminal half of E. coli RNase E is largely
unstructured and poorly conserved6. In E. coli and related proteobacteria, this Cterminal domain organizes and coordinates the activities of the multi-enzyme RNA
degradosome complex7,8, which is composed of enolase, polynucleotide
phosphorylase and the ATP-dependent helicase RhlB. The degradosome assembly is
required for the targeted degradation of transcripts9,10 and the coupled degradation of
small regulatory RNAs with their cognate transcript11.
Guided by the first structure for the RNase E/G family, a sequence alignment
was prepared for representative members of the group. The intricate details of the
structure account for the conservation of key residues at the catalytic site, the
hydrophobic core, and protein-protein interfaces, as indicated in Fig. S1.
A single protomer of RNase E catalytic domain bound to RNA occupies the
crystallographic asymmetric unit, and the tetramer is generated through crystal
symmetry. An oligomer of four identical subunits is consistent with our observations
from solution studies12. The tetramer is organized as a dimer-of-dimers with D2
symmetry. Shared zinc ions organize the two principal dimers. The location and
identification of the metal in the crystal structure was corroborated by its signal from
the anomalous scattering of X-ray (see Materials and Methods below). The presence
of thiol-linked zinc in the RNase E catalytic domain is in agreement with our solution
1
studies using X-ray spectroscopy13. In agreement with our mutagenic studies, the zinc
is coordinated at the intra-domain linker through a pair of cysteine residues in the
conserved CPxCxGxG motif that occurs throughout the RNase E/RNase G family13
(Fig. S1). A similar motif is found in the metal coordinating sites of the bacterial
chaperone DnaJ (PDB entry 1EXK), where a single zinc ion is coordinated within the
same subunit, rather than between two different subunits as in the case of RNase E.
The protomer can be dissected into a smaller and larger subdomain (Fig. S2a)
The principal dimer is formed mostly by the self-complementary interaction of the
larger domain of the protomer. The small domain also contributes to the principal
dimer through inter-subunit contact with the large domain. Another surface of the
small domain forms a self-complementary interface in the association of the two
principal dimers to create the tetramer.
The larger subdomain can be further subdivided structurally. Fig. S2 shows
the isolated subdomains of the RNase E catalytic domain. The RNase H fold is
discontinuous with regard to the linear sequence (residues 1-35 and 216-279) and is
interrupted by the insertion of two additional folds. The first embedded fold is an S1
domain, which is a widely occurring RNA-binding structural motif. The S1
subdomain of RNase E makes important contacts with the RNA near the cleavage
site. In comparison with the isolated S1 domain reported earlier14, there are small
adjustments in the positions of sidechains and exposed loops that can be attributed to
contacts to the RNA and other subdomains of RNase E. The DNase I domain
contains the catalytic site. Through self-association, this domain contributes the
greatest hydrophobic interface of the RNase E tetramer, and its dimer mimics DNase
I. Aside from its self-association, the small domain (residues 415 to 529) appears to
2
be structurally indivisible. The fold of this small domain is similar to that of a domain
of myo-inositol 1-phosphate synthase (1jki chain A, residues 323-422).
Mutations of the active site and 5’ sensing pocket
The RNA is not processed in the co-crystals studied here, probably because
the 2’-O-methyl substituent sterically occludes the strained transition state. The
crystal structure was used to identify residues of the catalytic site whose importance is
indicated by site direction mutagenesis (summarized in Table S1). These are
described in the main text. Phe67, Phe57 and Lys112 of the S1 domain form a basesequestering pocket, and substitution of any of these with Ala was found to abolish
catalytic activity of RNase E in vivo15. These observations corroborate the in vitro
observations of Diwa and Belasco16.
An analysis of mutations in the 5’-sensing pocket will be reported elsewhere
(Stead et al.,) The snug fit of the 5’ end into the pocket explains why RNA is
protected from cleavage by RNase E if this segment has a tri-phosphate cap or is
double-stranded: both forms are sterically excluded, as anticipated by Mackie17. The
sensing pocket differs from that of the PIWI domain used in RNA silencing
mechanisms18,19.
Cognate and non-cognate complexes
RNase E preferentially cleaves A+U-rich segments20-22 and it cuts the RNA Ibased substrate, 5’ACAGU*AUUUG, at the site indicated by the asterisk23. This
preferred cleavage site interacts with the catalytic magnesium in the complex of the
13-mer with RNase E (5’-pUUUACAGU*AUUUG). The separation between the 5’
sensor and the catalytic site suggests an explanation for the finding that RNase E does
3
not efficiently cleave bonds that are less than 8 nucleotides from the 5’monophosphated end of polyA or polyU substrates24,25. The 10-mer structure also has
the same optimal spacing between the 5’ sensing pocket and the catalytic magnesium.
As a consequence, the catalytic site of the 10-mer complex faces the sequence 5’pACAGUAUU#UG at the site marked by a hash sign (Fig. S3). The 15-mer spans
between the 5’ sensing pocket in a protomer of one tetramer to the catalytic site in the
protomer of in a symmetry related tetramer. It’s catalytic site faces the sequence 5’pUUUACAGUAUU#UGUU at the site marked by a hash sign. The protein-RNA
interactions for the 15-mer RNA nonetheless are similar to those seen for the 10-mer
and 13-mer. The 10-mer and 15-mer structures may represent non-cognate cleavage
complexes.
In all cases – both preferred and non-cognate complexes - the RNA follows
the same stacking arrangement over the surface of the RNA-binding channel, and
only one of the bases forms a hydrogen bond with the protein; Lys106 contacts the
exocyclic oxygen of the base at position -1 with regard to the scissile phosphate. This
direct contact may explain the preference for G or U at this position25. Aside from that
contact, there is no sequence recognition per se. We propose that the preference of
RNase E for A+U-rich substrates appears to arise mainly from “indirect readout”,
through the recognition of the RNA conformation. Thus, substrate preference is due to
the propensities of the bases to stack on the 5’ side of the scissile phosphate and then
pack at right angles immediately following the cleavage site, which is accommodated
more readily for A+U rich sequences.
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4
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