Direct localization of the tRNAs within the elongating ribosome by means of neutron scattering (proton-spin contrast-variation)

Structure of the elongating ribosome: Arrangement of the two tRNAs before and after translocation

“…For stabilization of the spin alignment the sample is kept at temperatures below 0.2 K. Both elongation states were measured two times for 1 week each. The functional integrity of the particles is not affected by this treatment as shown previously (7,9).…”

Structure of the elongating ribosome: Arrangement of the two tRNAs before and after translocation

“…For stabilization of the spin alignment the sample is kept at temperatures below 0.2 K. Both elongation states were measured two times for 1 week each. The functional integrity of the particles is not affected by this treatment as shown previously (7,9).…”

Protection Patterns of tRNAs Do Not Change during Ribosomal Translocation

“…On the other hand, it is known that the mRNA moves three nucleotides through the ribosome in the course of translocation (22), and also the mass centers of gravity of both the mRNA (23) and the two tRNAs move 12 Å within the ribosome (24), in good agreement with the length of one codon. Most important, the two tRNAs turn by an angle of at least 20°d uring translocation (25,26).…”

The E-site story: the importance of maintaining two tRNAs on the ribosome during protein synthesis

“…An analysis of formed polysomes in vivo revealed that at least 70% of the POST ribosomes carry an E-tRNA after an isolation procedure that lasts 15-20 h [28]. Similarly, POST ribosomes prepared in vitro can be pelleted through a sucrose cushion for 12 h without losing significant amounts of E-tRNA [29]. The important factor here is the use of in vivo near buffer conditions with low Mg 2+ (4.5-6 mM) and the presence of polyamines, particularly spermidine (2 mM).…”

Structure of the elongating ribosome: Arrangement of the two tRNAs before and after translocation

“…For stabilization of the spin alignment the sample is kept at temperatures below 0.2 K. Both elongation states were measured two times for 1 week each. The functional integrity of the particles is not affected by this treatment as shown previously (7,9).…”

Protection Patterns of tRNAs Do Not Change during Ribosomal Translocation

“…On the other hand, it is known that the mRNA moves three nucleotides through the ribosome in the course of translocation (22), and also the mass centers of gravity of both the mRNA (23) and the two tRNAs move 12 Å within the ribosome (24), in good agreement with the length of one codon. Most important, the two tRNAs turn by an angle of at least 20°d uring translocation (25,26).…”

The E-site story: the importance of maintaining two tRNAs on the ribosome during protein synthesis

“…An analysis of formed polysomes in vivo revealed that at least 70% of the POST ribosomes carry an E-tRNA after an isolation procedure that lasts 15-20 h [28]. Similarly, POST ribosomes prepared in vitro can be pelleted through a sucrose cushion for 12 h without losing significant amounts of E-tRNA [29]. The important factor here is the use of in vivo near buffer conditions with low Mg 2+ (4.5-6 mM) and the presence of polyamines, particularly spermidine (2 mM).…”

Structure of the elongating ribosome: Arrangement of the two tRNAs before and after translocation

“…For stabilization of the spin alignment the sample is kept at temperatures below 0.2 K. Both elongation states were measured two times for 1 week each. The functional integrity of the particles is not affected by this treatment as shown previously (7,9).…”

Protection Patterns of tRNAs Do Not Change during Ribosomal Translocation

“…On the other hand, it is known that the mRNA moves three nucleotides through the ribosome in the course of translocation (22), and also the mass centers of gravity of both the mRNA (23) and the two tRNAs move 12 Å within the ribosome (24), in good agreement with the length of one codon. Most important, the two tRNAs turn by an angle of at least 20°d uring translocation (25,26).…”

The E-site story: the importance of maintaining two tRNAs on the ribosome during protein synthesis

“…An analysis of formed polysomes in vivo revealed that at least 70% of the POST ribosomes carry an E-tRNA after an isolation procedure that lasts 15-20 h [28]. Similarly, POST ribosomes prepared in vitro can be pelleted through a sucrose cushion for 12 h without losing significant amounts of E-tRNA [29]. The important factor here is the use of in vivo near buffer conditions with low Mg 2+ (4.5-6 mM) and the presence of polyamines, particularly spermidine (2 mM).…”