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Figure 3.
Figure 3. In Vitro mRNA Cleavage Assay on the 70S Ribosome
(A) Sequence of E. coli RelE with the conservation among
homologs indicated as increasing strength of red color and the
conserved tyrosine at the C terminus in light blue. The
secondary structure is shown above the sequence and the
interactions to rRNA and mRNA below (all numbers correspond to
the E. coli 16S sequence). Residues in the P. horikoshii RelE
homolog that affect the activity are indicated with black boxes
(Takagi et al., 2005).
(B) Overview of the mRNAs used for
the in vitro cleavage assays. The 25 nt mRNA consists of a
Shine-Dalgarno element (SD) followed by a spacer and the P site
(AUG) and A site (UAG) codons. “Trunc Asite” ends after the
P site codon with a 3′-OH. The table shows predicted masses of
full length mRNA and fragments that would result from cleavage
after position 1 or 2 of the A site codon leaving either a
3′-OH, 3′-phosphate (3′-P), or 2′-3′ cyclic phosphate
(2′,3′-cP).
(C) MALDI mass spectrometry spectra and
masses of RNA fragments isolated from complexes in the absence
(blue) or presence (red) of RelE.
(D) In vitro cleavage
assay using 5′ ^32P-labeled mRNA substrates. • is the 25 nt
unmodified mRNA; MAP has phosphorothioate linkages after A site
codon positions 1 and 2; MAO, MAO2, and MAO3 are
2′-O-methylated at position 1, positions 1 + 2, or all three
positions, respectively; and MAD contains a deoxyribose at
position 1. The mRNAs were incubated with either T. thermophilus
(T.th.) or E. coli (E.co.) 70S ribosomes, tRNA^fMet, and either
RelE^wt or RelE^R81A/R45A (RelE^dm) as indicated for either 1 hr
(lanes 1–16) or overnight (lanes 17 and 18). The size markers
indicate the positions of the full-length (25 nt 3′-OH) and
Trunc Asite (18 nt 3′-OH) RNAs and the 20 nt 2′-3′ cyclic
phosphate cleavage product, which runs approximately 1 nt faster
than the corresponding 3′-OH species.
See also Figure S3.
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