Results: 5

1.
Fig. 1

Fig. 1. From: Dynamics of translation by single ribosomes through mRNA secondary structures.

mRNA designators used in this study and their secondary structures. Positions of mRNA structures are indicated, defining the 5′-base of the Arginine codon as +1 (mRNA sequences are in Supplementary Table 1). M, R, F, V, K, Y, and E are single-letter abbreviations for N-formylmethionine, Arginine, Phenylalanine, Valine, Lysine, Tyrosine, and Glutamate, respectively. Base pairs are indicated in blue, whereas unpaired bases are indicated in red. Boxes and arrows indicate the mutations. At 23 °C, the most stable structure of mPL (ΔG = –5.6 kcal/mol, Supplementary Table 1) is far less stable than the structures of mPK-SL (ΔG = –37.1 kcal/mol) and mSL-15 (ΔG = –42.6 kcal/mol).

Chunlai Chen, et al. Nat Struct Mol Biol. 2013 May;20(5):582-588.
2.
Fig. 5

Fig. 5. From: Dynamics of translation by single ribosomes through mRNA secondary structures.

L1-tRNA smFRET. (AC) Typical real-time ribosome translation traces measured using Cy5-L1 and 10 nM Cy3-R TC with mRNA mPL in the presence of 50 nM F-TC (A), 5 nM F-TC (B), and mRNA mPK at 50 nM F-TC (C). Traces and recording conditions are as in Fig. 4. (D) Dwell time distributions of POSTRF complexes (complex 6) from L1-tRNA measurements. Unlabeled TC concentrations were 50 nM unless indicated. (E) Apparent dissociation rates of Cy3-R from the E-site were calculated from single exponential fitting (black curves) of dwell time of POSTRF complexes (D). Error bars represent S.E.M., n ≥ 129.

Chunlai Chen, et al. Nat Struct Mol Biol. 2013 May;20(5):582-588.
3.
Fig. 4

Fig. 4. From: Dynamics of translation by single ribosomes through mRNA secondary structures.

L11-tRNA smFRET. (A and B) Typical real-time ribosome translation traces measured using Cy3-R and Cy5-L11 (A) or Cy3-F and Cy5-L11 (B), for ribosomes programmed with mPL in the presence of 2 μM EF-G·GTP. Cy3 fluorescence (green) and sensitized emission of Cy5 (FRET, blue) under 532 nm excitation were collected. (C and D) Dwell time distributions of PREER complexes (C; complex 2) and PRERF complexes (D; complex 5) from L11-tRNA measurements. (E and F) Apparent translocation rates of PREER (E) and PRERF (F) complexes, respectively, from single exponential fitting (black curves) of dwell times of PREER (C) and PRERF (D). Camera settings and abbreviations for mRNAs are as in Fig. 3. Error bars represent S.E.M., n ≥ 181.

Chunlai Chen, et al. Nat Struct Mol Biol. 2013 May;20(5):582-588.
4.
Fig. 2

Fig. 2. From: Dynamics of translation by single ribosomes through mRNA secondary structures.

FRET studies on complexes formed through two elongation cycles that add Arginine and Phenylalanine to the nascent peptide. (A) Names and numerical designations of complexes. PRE and POST stand for pre-translocation and post-translocation complexes, respectively. E, R, and F are single-letter abbreviations for Glutamate, Arginine, and Phenylalanine, respectively. L11 and L1 are two large subunit ribosomal proteins. E, P, and A are the three tRNA binding sites on the ribosome. (BE) smFRET experiments employed: Cy3-labeled Arg-tRNAArg (Cy3-R) and Cy5-labeled Phe-tRNAPhe (Cy5-F) (B); Cy3-R or Cy3-labeled Phe-tRNAPhe (Cy3-F) and Cy5-L11 (C and D); Cy3-R and Cy5-L1 (E). Labeled green and red dots indicate Cy3- and Cy5-labeled components, respectively. The thickness of the blue halo around the red dots qualitatively indicates the expected FRET efficiency.

Chunlai Chen, et al. Nat Struct Mol Biol. 2013 May;20(5):582-588.
5.
Fig. 3

Fig. 3. From: Dynamics of translation by single ribosomes through mRNA secondary structures.

tRNA-tRNA smFRET. (A) Typical real-time ribosome translation trace measured using Cy3-R and Cy5-F, for ribosomes programmed with mPL. In addition to Cy3 fluorescence (green) and sensitized emission of Cy5 (FRET, blue) under 532 nm excitation, Cy5 direct fluorescence (magenta) was collected under alternating 640 nm laser excitation. Camera recording was started 10 seconds prior to injection of 10 nM Cy3-R and Cy5-F ternary complexes, 50 nM unlabeled Y and E ternary complexes, 2 μM EF-G, and 2 mM GTP and continued for 10 minutes at 100 ms integration time per frame. (B and C) Dwell time distributions of ribosome complexes programmed with different mRNAs (mPL, mSL-15, mPK) containing either Cy3-R only (B, complexes 2–4) or Cy3-R and Cy5-F, giving rise to FRET (C, complexes 5 and 6), respectively, fitted with single exponential decay curves (black lines), to give apparent rate constants for conversion of PREER (complex 2) to PRERF (complex 5) and of PRERF (complex 5) to POSTF (complex 7), respectively. In each case, times were collected from 200-400 ribosomes. (D and E) Apparent rate constants for steps from PREER to PRERF (D) and from PRERF to POSTF (E) for ribosomes programmed with the indicated mRNAs (including mPK-G3C G4C (3 4) and mPK-G15C G42C (15 42)). Error bars represent S.E.M., n ≥ 173.

Chunlai Chen, et al. Nat Struct Mol Biol. 2013 May;20(5):582-588.

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