Versatility of Trigger Factor Interactions with Ribosome-Nascent

Supplemental Data

Versatility of Trigger Factor Interactions with Ribosome-Nascent

Chain Complexes

Sathish Kumar Lakshmipathy, Rashmi Gupta, Stefan Pinkert, Stephanie Anne Etchells and F. Ulrich Hartl

Table of ContentsPage

Supplemental Experimental Procedures2

Supplemental Figures S1-S53-7

Supplemental Tables S1-S38-9

Supplemental References10

Supplemental Experimental Procedures

Ribosome binding assay

Ribosomes purified fromE. coli MC4100 were incubated with 1 µM TF, TF-B, TF150-NBD, TF326-NBD or TF376-NBD for 10 min in CTF buffer (50 mM triethanol amine pH 7.5, 50 mM KOAc, 5 mM Mg(OAc)2, 1 mM DTT)at 30 °C. Following incubation, the reaction was layered over a sucrose cushion (0.5 M sucrose (w/v), 14 mM Mg(OAc)2, 120 mM KOAc and 20 mM HEPES pH 7.5) and centrifuged for 20 min at 100, 000 rpm (4 °C). The resulting supernatant was removed and precipitated with an equal volume of 50% trichloroacetic acid (v/v), and washed once with 1 ml of 100% acetone and resuspended in SDS sample buffer. The pellet from the sucrose cushion was resuspended directly in SDS sample buffer prior to separation on SDS-PAGE.

Fluorescence change of TF variants upon ribosome binding

1 µM TF-B, TF150-NBD, TF326-NBD or TF376-NBD was incubated for 10 min in the presence or absence of 1 µM ribosomes in CTF buffer at 30 ºC. Following incubation a fluorescence emission scan was recorded with excitation at 387 nm for TF-B or 472 nm for TF-NBD.

Supplemental Figures

Figure S1

Supplemental Figure S1

Labeled and unlabeled TF binds to ribosomes with similar efficiency

(A) Binding of TF and TF-B to purified E. coli ribosomes. TF and TF-B were incubated alone or with purified ribosomes, followed by centrifugation and analysis by SDS-PAGE and Coomassie blue staining (see Supplemental Experimental Procedures). S, supernatant; P, pellet fraction. TF and TF-B both appeared in the pellet fraction (lanes 8 and 10) in the presence of ribosomes in a ~1:1 ratio relative to ribosomal protein S1. The position of TF and S1 is indicated with an arrow.

(B and C) TF150-NBD, TF326-NBD and TF376-NBD were analyzed as in (A) in the presence of ribosomes (B) or the absence of ribosomes (C). All the labeled TF proteins sedimented with the ribosomes in a ~1:1 ratio similar to unlabeled TF.A minor fraction of TF is found in the pellet fraction in the absence of ribosomes. This could be due to aggregation of TF during incubation or during the centrifugation at 100, 000 rpm.

Figure S2

Supplemental Figure S2

Fluorescence emission scans of TF variants in the presence or absence of ribosomes

(A) Emission scans of TF-B in the presence (black circles) or absence of purified E. coli ribosomes (red circles). A decrease of ~40% in emission intensity was observed due to ribosome binding (1,2).

(B) Emission scans of TF150-NBD, TF326-NBD or TF376-NBD in the absence (red circles, black triangles, or red diamonds, respectively) or presence (purple squares, green triangles or grey hexagons, respectively) of purified E. coli ribosomes. No significant change in fluorescence was observed in the presence or absence of ribosomes for TF labeled with NBD.

Figure S3

Supplemental Figure S3

Hydrophobicity scores of the predicted strong TF interactors and Luc

Bar graphs representing the hydrophobicity scores for the continuous hydrophobic segments of the predicted strong interactors used in this study (left) and for Luc (right) (see Figure 2A). A minimum of 5 consecutive residues in the central region of a 15 amino acid window having a mean hydrophobicity value of < −0.5 kcal/mol was considered a potential motif for TF binding(1). The hydrophobicity score is defined as the number of central amino acids that are part of such hydrophobic motifs (see Experimental Procedures). RIMK has one hydrophobic region with the score of +18 between residues 1-19. AROE [+6 (residues 199-205), +12 (residues 241-253)], DCP [+6 (residues 275-281), +15 (residues 601-616)], and GATD [+8 (residues 82-90), +13 (residues 166-179)] each have two hydrophobic regions (1 and 2), respectively. Luc has five hydrophobic regions 1 to 5 with scores of +13 (residues 87-100), +7 (residues 238-245), +6 (residues 248-254), +5 (residues 255-260), and +6 (residues 285-291), respectively.

Figure S4

Supplemental Figure S4

Fractional amplitudes corresponding to fast and slow phases of dissociation of TF326-NBD and TF376-NBD from full-length LucRNCs

Fractional amplitudes corresponding to fast (black) and slow phases (grey) are indicated in percent. Standard deviations from three independent experiments are shown. Also see Figure 3.

Figure S5

Supplemental Figure S5

Translation of the predicted weak interactors and predicted strong interactors of TF

Translation of the predicted weak (left) and strong interactors (right) in the presence of 35S-methionine in the PURE system. Translation ofstalled nascent chains was initiated by the addition of DNA and performed for ~50 min. Samples were loaded on SDS-PAGE followed by autoradiography. Black arrows indicate hydrolyzed nascent chains and arrowheads indicate peptidyl-tRNAs.

Supplemental Tables

Table S1: Half-time (t1/2values) of TF-NBD dissociation from various Luc RNCs

Nascent
chain / Length of nascent chain / TF present during translation / Excess TF added as competitor / Fast t1/2
value / Slow t1/2
value
Luc / Full-length / TF326-NBD / TF / 14 ± 2 s / 102 ± 16 s
Luc / 164 mer / TF326-NBD / TF / 18 ± 3.5 s
Luc / 550 mer / TF326-NBD / TF / 14 ± 3 s / 77 ± 23 s
Luc / Full-length / TF376-NBD / TF / 8 ± 2 s / 88± 23 s
Luc / 164 mer / TF376-NBD / TF / 10 ± 1.5 s
Luc / 550 mer / TF376-NBD / TF / 10 ± 1 s / 95 ± 12 s
Luc / Full-length / TF150-NBD / TF / 111 ± 7 s
Luc / 550 mer / TF150-NBD / TF / 102 ± 4 s
Luc / Full-length / TF326-NBD / TF(FRK/AAA) / No competition / No competition

Data from competition of labeled TF with unlabeled TF were used to calculate the half-time (t1/2 values) of TF-NBD dissociation from various Luc RNCs. The data were analyzed using a three parameter, single exponential function or a five parameter, double exponential function as described in Experimental Procedures (Also see Figure 3).

Table S2: Representatives of predicted strong and weak interactors of TF

Predicted strong interactors / Predicted weak interactors
Dipeptidyl carboxypeptidase II (DCP) / ATP phosphoribosyltransferase (HISG)
Shikimate dehydrogenase (AROE) / Subunit of glutamyl-tRNA reductase (HEMA)
Ribosomal protein S6 modification protein (RIMK) / GRPE
Galactitol-1-phosphate dehydrogenase (GATD) / Methionine adenosyltransferase (METK)
5,10-Methylenetetrahydrofolate reductase (METF)
Subunit of deoxyguanylate kinase/guanylate kinase (SPOR)
Ribosome recycling factor (RRF)
30S ribosomal protein S7 (RPSG)
23S rRNA m2G1835 Methyltransferase (RLMG)

The proteins analyzed were amplified from E. coli genomic DNA without the terminal stop codon. The regulatory components for in vitro translation such as the promoter for T7 RNA polymerase and the ribosomal binding site were added using the universal primer supplied with the PURE system kit.

Table S3: Half-time (t1/2 values) of TF326-NBD dissociation from RNCs of predicted weak and strong interactors

Nascent
Chain / TF present during translation / Excess TF added as competitor / t1/2 value
SPOR / TF326-NBD / TF / 13.5 ± 1 s
AROE / TF326-NBD / TF / 9.5 ± 0.5 s
RIMK / TF326-NBD / TF / 10 ± 2.5 s
GATD / TF326-NBD / TF / 24.5 ± 1 s
DCP / TF326-NBD / TF / 28.5 ± 6 s

Data from competition experiments were used to calculate the half-time (t1/2 values) of TF326-NBD dissociation from the RNCs of predicted weak and strong interactors. The data were analyzed using a three parameter, single exponential function as described in Experimental Procedures (also see Figure 5).

Supplemental References

1.Kaiser, C. M., Chang, H. C., Agashe, V. R., Lakshmipathy, S. K., Etchells, S. A., Hayer-Hartl, M., Hartl, F. U., and Barral, J. M. (2006) Nature444, 455-460