Ribosome binding by tRNAs with fluorescent labeled 3′ termini

Abstract

Yeast and E. coli tRNAPhe samples were oxidized and labeled at the 3' end with dansyl hydrazine or fluorescein thiosemicarbazide. These tRNAs can bind to poly(U)-programmed E. coli 70S tight couple ribosomes in 25 mM magnesium at 8 degrees C. Two binding sites with binding constants of about 1 X 10(9) M-1 (P) and 3 X 10(7) M-1 (A) were determined for the yeast tRNAPhe derivatives. With E. coli tRNAPhe the A site affinity is similar to yeast tRNAPhe but the P site affinity is 5-fold weaker. Singlet-singlet energy transfer showd that the distance from the 3' end of tRNAPhe in the P site to a fluorescein derivative of erythromycin is 23 A. This supports in vitro studies suggesting that erythromycin binds near the peptide moiety of peptidyl tRNA. A distance of 34 A between the 3' ends of 2 tRNAs bound simulatneously on the ribosome was also measured. This long distance may mean that the deacylated fluorescent tRNA binds to the A site in an orientation like that in the stringent response rather than in protein synthesis.

Effect of ribosome binding and translocation on the anticodon of tRNAPheas studied by wybutine fluorescence

“…1 do not depend upon aminoacylation of the tRNA since identical spectra have been observed for the A or P-site complexes of non-aminoacylated tRNAPhe (not shown). As far as P-site binding is concerned, these results are very similar to spectral data obtained previously (21), whereas the present A-site spectrum is rather different from the one reported by these authors. We feel that our spectrum, which has been measured directly with the isolated A-site complex, is more reliable than the previously reported one, which was obtained by decomposing a mixed spectrum of free, P site-bound, and A site-bound tRNAPhe on the basis of relative concentrations calculated from the binding constants.…”

Measurements of Internal Distance Changes of the 30S Ribosome Using FRET with Multiple Donor–Acceptor Pairs: Quantitative Spectroscopic Methods

“…This value has been used previously in the literature for the ribosome and molecular environments containing similar high RNA and protein content. [19][20][21]44,45 This value is expected to represent an average of contributions from solvent as well as the molecule itself (for our solvent nZ1.34, measured using an Abbe refractometer (Bausch and Lombe, Rochester, NY), for nucleic acids it has been found that nw1.75). 46 This value is different from the refractive index of the bulk solution, which is used for the quantum yield The lower integration limits of the donor and acceptor conjugates were 420 nm and 450 nm, respectively.…”

Calculation of the relative geometry of tRNAs in the ribosome from directed hydroxyl-radical probing data

“…Strand scission of tRNAs by hydroxyl radicals generated from 59-Fe(II)-tRNA+ tRNA f Met , complexes with tRNA f Met [ 32 P]pCp bound to the P site, and either tRNA Phe (GMP) or 59-Fe(II)-tRNA Phe (GMPS-Fe) bound to the A site+ tRNA Lys , complexes with tRNA Lys [ 32 P]pCp bound to the A site, and either tRNA Phe (GMP) or 59-Fe(II)-tRNA Phe (GMPS-Fe) bound to the P site+ Lanes U2, Phy M, and T1 are enzymatic sequencing of tRNA[ 32 P]pCp using the respective ribonucleases; Ak: alkaline hydrolysis of tRNA[ 32 P]pCp; Mock: reaction chemically treated as for the probing reaction, but in the absence of ribosome+ Positions of strand scission are indicated by the bars+ of the two tRNAs+ These two mutually exclusive arrangements have been designated as the R and S orientations, respectively (Lim et al+, 1992)+ Fluorescence resonance energy transfer experiments provided important constraints for modeling the mutual arrangement of tRNAs (Fairclough & Cantor, 1979b;Wells & Cantor, 1980;Johnson et al+, 1982;Paulsen et al+, 1983) + Paulsen et al+ (1983) proposed a model with a 60 6 308 angle between the A-and P-site tRNAs, but were unable to distinguish between the R and S orientations using FRET constraints+ Although the FRET data by themselves were insufficient to decide this issue, they served the useful purpose of eliminating models that were inconsistent with the measured distances+ Based on a stereochemical analysis of the peptidyl transferase reaction, Spirin and Lim (1986) proposed Figure 5+ an R orientation with a 1008 angle between the two tRNAs+ Ofengand et al+ (1986), based on tRNA crosslinks to 30S subunit protein S19 and other biochemical data, proposed a model with the tRNAs in the S orientation with an angle of 658 between the tRNA planes+ A detailed stereochemical model using the crystal structure of tRNA and incorporating the FRET data was proposed by McDonald and Rein (1987), who maintained the tRNA crystal structures as rigid bodies and manipulated the conformation of the mRNA+ Their model has an S orientation with an approximately 458 angle between the tRNA planes and a 338 kink between the two mRNA codons+ Models with mRNA fixed in an idealized A-form geometry, deforming the anticodon loops of the tRNAs from the crystal structure (Prabahakaran & Harvey, 1989), or where both the mRNA codons and tRNAanticodonloopshavealteredconformations (Easterwood et al+, 1994) were also proposed; the tRNAs in these models are in the S orientation with an angle of approximately 458+ While the S orientation is preferred in some of the models based on crosslinking and chemical protection studies (Stern et al+, 1988;Wower et al+, 1989Wower et al+, , 1993Noller et al+, 1990;Nagano et al+, 1991;Nagano & Nagano, 1997), Lim and coworkers invoked extensive tRNA, mRNA, and rRNA crosslinking data to derive a model with R ...…”