Alanine scanning and Fe-BABE probing of the bacteriophage ø29 prohead RNA-connector interaction

Abstract

The DNA packaging motor of the Bacillus subtilis bacteriophage ø29 prohead is comprised in part of an oligomeric ring of 174 base RNA molecules (pRNA) positioned near the N termini of subunits of the dodecameric head-tail connector. Deletion and alanine substitution mutants in the connector protein (gp10) N terminus were assembled into proheads in Escherichia coli and the particles tested for pRNA binding and DNA-gp3 packaging in vitro. The basic amino acid residues RKR at positions 3-5 of the gp10 N terminus were central to pRNA binding during assembly of an active DNA packaging motor. Conjugation of iron(S)-1-(p-bromoacetamidobenzyl) ethylenediaminetetraacetate (Fe-BABE) to residue S170C in the narrow end of the connector, near the N terminus, permitted hydroxyl radical probing of bound [(32)P]pRNA and identified two discrete sites proximal to this residue: the C-helix at the junction of the A, C and D helices, and the E helix and the CE loop/D loop of the intermolecular base pairing site.

Figure 1

(a) ø29 pRNA secondary structure determined by mfold with stem/loop letter designations (blue). Residues involved in intermolecular base pairing are boxed. (b) N-terminal residues (multi-colors), disordered in the connector crystal structure, modeled onto the connector structure (gray). Residues R3, K4, and R5 are implicated in the binding of pRNA. Residue S170 conjugated with Fe-BABE was used in the mapping of the connector-pRNA interaction. (c) Cryo-EM density fitted with the crystal structure of the dodecameric connector (yellow) displays the spacial relationship of the N-termini of the connector, density interpreted as a pentameric pRNA (green) forming a basket around the connector, and density interpreted as capsid (gray) appearing to contact the pRNA. The circular inset shows the close proximity of the N-termini of connector subunits (beginning with residue 11 and denoted by orange stars) and residues S170 (marked by blue dots) to the density of the pRNA. Ribbon model of the pRNA (magenta) is superimposed onto spokes of density interpreted as the pRNA A helix.

Figure 2

Connector protein N-terminus sequence alignments of ø29 and relatives by T-coffee. Magenta dots indicate residues that are disordered in the N-termini of subunits of the ø29 connector crystal structure, and the helix denotes amino acids resolved in helix 1 of the ø29 connector. Identical residues are indicated by a star and semi-conserved residues by a colon.

Figure 3

EMSA analysis of pRNA binding to proheads containing mutations of the ø29 connector protein N-terminus. [³²P] 5’ end-labeled 120nt pRNA was incubated with proheads containing wild-type, deletion, and alanine-substituted mutations of the connector N-terminus, and the complexes were detected by agarose gel electrophoresis, followed by autoradiography. [³²P] pRNA bound to particles is observed near the top of the gel, whereas free [³²P] pRNA is found near the bottom of the gel. The first letter of mutant designations indicates the wild-type amino acid residue that was mutated to alanine, and the number denotes its position in the connector protein N-terminal amino acid sequence. Lanes 1 and 12 show the position of free pRNA in the absence of particles.

Figure 4

In vitro DNA packaging by ø29 proheads containing wild-type, deletion, or alanine-substituted mutations in the connector protein N-terminus. The first letter indicates the wild-type amino acid residue that was mutated to alanine, and the number denotes its position in the amino acid sequence. Lanes 1 and 2 show 50% and 100% of the input DNA added to the reaction, respectively, for quantification of DNA packaging. DNA in lanes 3-12 represent packaged DNA that is protected from DNaseI treatment (see Materials and Methods). Negative controls in which ATP is omitted from the packaging reaction were done for each particle type. One such control is shown in lane 13.

Figure 5

SDS-PAGE showing the amount of gp10 assembled into cys- proheads versus proheads with an N-terminal cysteine insertion or with N-terminal cysteine substitutions. The other prohead constituents are major head protein (gp8), head-fiber protein (gp8.5) and scaffolding protein (gp7).

Figure 6

SDS-PAGE of proheads showing Cy3-maleimide labeling of particles containing gp10 without the native cysteines, particles having gp10 with a cysteine insertion, or particles having gp10 with substitutions before (-) and after (+) conjugation of the connectors with Fe-BABE.

Figure 7

Hydroxyl radical cleavage pattern of [³²P]120nt pRNA bound to proheads with Fe-BABE-conjugated connectors having the R3C, R5C or S170C mutations, compared to cleavage by mock-conjugated cys- proheads. Arrows point to the cleavage locations in the pRNA secondary structure, and residues shown in magenta are the specific sites of cleavage. Nucleotides protected by the prohead from ribonuclease digestion are enclosed in the gray boxes. Bases 1 through 25 showed no distinct cleavage patterns (data not shown).