Linet al.,Science 367 , 100–104 (2020) 3 January 2020 2of5
Fig. 1. TTC5 interacts with the
N termini of nascent tubulins.
(A) Experimental strategy to detect
interaction partners close to the
N terminus of nascent tubulin. The
UV-activated cross-linking amino acid
p-benzoyl-L-phenylalanine (Bpa) is
introduced site-specifically at position 7
using amber suppression (see fig. S1).
IVT, in vitro translation; XL, cross-linking;
PAGE, polyacrylamide gel electrophoresis.
(B)Photo–cross-linking analysis of
(^35) S-labeled 94–amino acid ribosome–
nascent chain complexes (RNCs) of
humanb-tubulin and mutants (indicated
in red) in the N-terminal MREI motif.
The positions of non–cross-linked tRNA-
associated nascent chain (NC-tRNA)
and a cross-linking partner specific to
wild-type tubulin (red asterisk) are
indicated. Other nascent chain cross-links
agnostic to the MREI motif are indicated
by black asterisks. (C) Quantitative
mass spectrometry of proteins copurified
with wild-type (WT) versus MHQV
mutant (mut)b-tubulin RNCs plotted
by molecular weight. (D)Photo–cross-
linking and immunoprecipitation (IP)
analysis of^35 S-labeled 94–amino acid RNCs ofa-orb-tubulin compared to the indicated mutants. Amino acid abbreviations in this or later figures: A, Ala; C, Cys; D, Asp;
E, Glu; F, Phe; G, Gly; H, His; I, Ile;K, Lys; M, Met; Q, Gln; R, Arg; V, Val.
Fig. 2. Mechanism of ribosome–nascent
chain engagement by TTC5.(A) Overview
of the cryo-EM–derived structure of the
complex between TTC5 and a ribosome
containing the first 64 amino acids ofb-tubulin.
(B) Close-up view of the TTC5 interaction
with 28SrRNA. Three conserved Lys residues
in TTC5 within electrostatic interaction distance
of the rRNA backbone are indicated. (C) The
surface of TTC5 that interacts with ribosomal
protein uL24 is indicated in orange. The
28 S-interacting residues from (B) are shown
in yellow. The N-terminal eight amino acids
of theb-tubulin nascent chain are shown in red
within the chain’s binding groove of TTC5.
(D) Close-up view of the N-terminal eight amino
acids of nascentb-tubulin (MREIVHIQ) within
TTC5. Yellow spheres denote C-batoms for the
indicated side chains (not modeled). Arg^147 is
within salt-bridge distance of Glu^3 , and Asp^225
and Glu^259 are within salt-bridge distance of
Arg^2. Phe^194 on the“floor”of the binding
groove, shown in Fig. 3A to cross-link with
nascentb-tubulin, is indicated.
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