Features of 80S mammalian ribosome and its subunits

Abstract

It is generally believed that basic features of ribosomal functions are universally valid, but a systematic test still stands out for higher eukaryotic 80S ribosomes. Here we report: (i) differences in tRNA and mRNA binding capabilities of eukaryotic and bacterial ribosomes and their subunits. Eukaryotic 40S subunits bind mRNA exclusively in the presence of cognate tRNA, whereas bacterial 30S do bind mRNA already in the absence of tRNA. 80S ribosomes bind mRNA efficiently in the absence of tRNA. In contrast, bacterial 70S interact with mRNA more productively in the presence rather than in the absence of tRNA. (ii) States of initiation (P(i)), pre-translocation (PRE) and post-translocation (POST) of the ribosome were checked and no significant functional differences to the prokaryotic counterpart were observed including the reciprocal linkage between A and E sites. (iii) Eukaryotic ribosomes bind tetracycline with an affinity 15 times lower than that of bacterial ribosomes (K(d) 30 microM and 1-2 microM, respectively). The drug does not effect enzymatic A-site occupation of 80S ribosomes in contrast to non-enzymatic tRNA binding to the A-site. Both observations explain the relative resistance of eukaryotic ribosomes to this antibiotic.

Figure 1.

Sucrose gradient profiles of small ribosomal subunits and ribosomes (A₂₆₀) in the presence of [³²P]mRNA (dotted lines) and—where indicated—tRNA^Phe (dashed line in 1b represents experiment in the presence of non-cognate tRNA^Ala). Panels on the left and right sides show profiles obtained with eukaryotic and bacterial ribosomes, respectively.

Figure 2.

AcPhe-tRNA binding (red lines) and puromycin reaction (black lines) with 80S ribosomes in the presence (a) and absence (b) of MFVK-mRNA. ν, tRNA bound per ribosome in a binding assay or acylated tRNA per ribosome reacting with puromycin.

Figure 3.

Addition of a ternary complexes (green) to a P_i state of 80S ribosomes. (a) Binding of the ternary complex eEF1A·GTP·Val-tRNA to 80S ribosomes in the P_i state carrying an AcPhe-tRNA (red line) at the P site in the presence of MFVK-mRNA. (b) Di-peptide formation in the assay shown in (a). (c) eEF1A·GDPNP·Val-tRNA (green bars) is added to P_i states carrying AcPhe-tRNA (red bars). ν, tRNA bound per ribosome in a binding assay or acylated tRNA per ribosome reacting with puromycin.

Figure 4.

Binding of the ternary complex eEF1A·GTP·[¹⁴C]Lys-tRNA (green) to 80S ribosomes in the POST state carrying an Ac[³H]Val-tRNA (red) at the P and a [³²P]tRNA^Phe (orange) at the E site. The 100% binding corresponds to 0.4 tRNA^Phe bound per ribosome (ν). For details see text.

Figure 5.

Analysis of tetracycline binding to 70S and 80S ribosomes. (a) Binding of [³H]Tet to empty 70S (diamonds, dashed line) and 80S (circles, solid line) ribosomes. (b) Poly(U)-poly(Phe) synthesis in bacterial (diamonds, dashed line) and mammalian (circles, solid line) systems in the presence of increasing amounts of tetracycline. The 100% corresponds to 60 and 15 incorporated Phe per ribosome in the bacterial and eukaryotic system, respectively. (c) Effect of tetracycline on non-enzymatic A-site binding with AcPhe-tRNA (blue) and enzymatic A-site binding with ternary complex (red). Deacylated tRNA^fMet and were used to occupy the P site in 70S (dashed line) and 80S systems (solid line), respectively. The 100% corresponds to a ν value of 0.7 for both 70S and 80S ribosomes for non-enzymatic A-site binding and 0.83 and 0.6 for 70S and 80S ribosomes, respectively, for enzymatic A-site binding. (d) Parts of helices 34 from phylogenetic conservation maps superimposed onto the bacterial (E. coli), archaeal (Methanococcus jannaschii) and eukaryotic (Saccharomyces cerevisiae) 16S-type secondary structures (http://www.rna.ccbb.utexas.edu/). ACGU, 98+% conserved positions; acgu, 90–98% conserved positions; filled circle, 80–90% conserved positions; open circle, less than 80% conserved positions. The nucleotides of the right strand are connected for the sake of clarity; number of sequences considered: Bacteria, 4214; Archaea, 174; Eukaryota, 1939.