Binding of tRNA alters the chemical accessibility of nucleotides within the large ribosomal RNAs ofE. coliribosomes

Abstract

Functionally active 70S ribosomes were chemically modified with dimethylsulfate (DMS) in the presence and absence of bound tRNA. The ribosomal 16S RNA and 23S RNA were extracted, separated and labeled radioactively at their 3'-ends. DMS modification sites within the last 200 nucleotides from the 3'-ends were investigated on sequencing gels, after borohydride reduction and aniline catalyzed strand scission of the isolated RNA's. tRNA binding caused enhanced reactivity at 9 nucleotide positions while three sites showed decreased reactivity in the 16S RNA. The effects of bound tRNA on the modification of 23S RNA were limited. Only one enhancement was observed in the presence of bound tRNA. mRNA binding alone showed two more sites with enhanced reactivity, however. The results are consistent with the view that the sequence 1400-1500 of the 16S RNA plays an important functional role in the translating ribosome and possibly constitutes part of the tRNA binding site.

tRNA binding modifies the properties of the small ribosomal subunit of rat liver

“…The differences with our results, which clearly show that tRNA is able to convert pratically all the dimers into monomers without the addition of any factor, must be related to the different experimental conditions used in these studies. Concerning the mRNA-independent tRNA binding which we demonstrated, it is interesting to note that this also occurs on E. Coli ribosome inducing the same conformational change of ribosome-bound rRNAs as that observed when the messenger is present (17,18).…”

“…Therefore, the most likely hypothesis is that tRNA binding induces modifications of proteinrRNA interactions in large domains of the subunit. It should be noted that tRNA binding on E. Coli ribosomes induces a conformational change of rRNAs as measured by chemical accessibility of nucleotides (17,18). Our results give the first indications on extensive effects of tRNA binding on both mammalian large (3) and small ribosomal subunits.…”

Structure and function of ribosomal RNA

“…in the presence of tRNA, or in 70S ribosomes as opposed to isolated subunits) must be interpreted with caution. This has recently been made very clear by the experiments of Meier & Wagner (1984, 1985 who found that some of the guanine residues in E. coli 16S RNA that were protected from kethoxal modification in 70S ribosomes or polysomes (Brow & Noller, 1983) actually showed an enhanced reactivity to dimethyl sulphate. Since dimethyl sulphate reacts with N-7 of guanine, whereas kethoxal binds to the opposite side of the purine ring, this result shows that the observed effects are due to a rotation of the guanine residue concerned, rather than to a 'bulk shielding' by another ribosomal component.…”

“…The effects of tRNA binding or subunit association on the accessibility of individual residues in the RNA have also been reported (e.g. Meier & Wagner, 1984, 1985. These experiments have been confined to a study of the terminal regions of the rRNA molecules concerned, since they rely on end-labelling techniques and sequencing gels for the analysis of the modified or cleaved RNA sites and in consequence are limited by the resolving power of the sequencing gels (approx.…”

Analysis of a sequence region of 5S RNA fromE. colicross-linkedin situto the ribosomal protein L25

“…The ribosomes were precipitated with 0.67 volumes ethanol (-20°C, 1 hour), and the pellets were washed with 70% ethanol. 5S RNA isolation Total ribosomal RNA was isolated by phenol extraction as described in [17] and the individual RNAs were separated on 5%…”