Function of the ribosomal E-site: a mutagenesis study - PubMed (original) (raw)

Function of the ribosomal E-site: a mutagenesis study

Petr V Sergiev et al. Nucleic Acids Res. 2005.

Abstract

Ribosomes synthesize proteins according to the information encoded in mRNA. During this process, both the incoming amino acid and the nascent peptide are bound to tRNA molecules. Three binding sites for tRNA in the ribosome are known: the A-site for aminoacyl-tRNA, the P-site for peptidyl-tRNA and the E-site for the deacylated tRNA leaving the ribosome. Here, we present a study of Escherichia coli ribosomes with the E-site binding destabilized by mutation C2394G of the 23S rRNA. Expression of the mutant 23S rRNA in vivo caused increased frameshifting and stop codon readthrough. The progression of these ribosomes through the ribosomal elongation cycle in vitro reveals ejection of deacylated tRNA during the translocation step or shortly after. E-site compromised ribosomes can undergo translocation, although in some cases it is less efficient and results in a frameshift. The mutation affects formation of the P/E hybrid site and leads to a loss of stimulation of the multiple turnover GTPase activity of EF-G by deacylated tRNA bound to the ribosome.

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Figures

Figure 1

Figure 1

Binding of deacylated tRNAPhe to ribosomes. The graph shows the number of tRNA molecules bound to wild-type ribosomes (A), or to ribosomes carrying the C2394G mutation (B), versus tRNA excess over ribosomes. The curves are marked according to the presence or absence of a polyU template and to the tRNA molecule used. Closed triangles indicate ribosomes with presence of tRNA and absence of polyU. Closed diamonds indicate ribosomes with presence of both tRNA and polyU. Closed squares correspond to ribosomes with presence of polyU and tRNA-Δ3′A. ‘tRNA’ corresponds to intact tRNAPhe, ‘tRNA-Δ3′A’ corresponds to tRNAPhe lacking the 3′-terminal adenine.

Figure 2

Figure 2

Stepwise translation of the MFK-encoding mRNA analogue. (A) Partial sequence of the mRNA analogue. The Shine–Dalgarno sequence, the codons being translated and the corresponding toe-print stops are marked. The toe-print stop is repeatedly appearing at the nucleotide +16 relative to the 5′-most nucleotide at the P-site. (B) Toe-print analysis of the stepwise translation progress. ‘WT’ and ‘C2394G’ mark the complexes formed by wild-type ribosomes and ribosomes carrying the C2394G mutation, respectively. Lane 1, complex of ribosomes (0.1 µM), MFK-mRNA (0.1 µM) and tRNAfMet (0.2 µM). Lane 2, ribosomes (0.1 µM), MFK-mRNA (0.1 µM), tRNAfMet (0.2 µM) and AcPhe-tRNAPhe (0.2 µM). Lane 3, same as in lane 2 but after the addition of EF-G*GTP (0.1 µM). Lane 4, corresponds to lane 3 but with Lys-tRNALys*EF-Tu*GTP (0.2 µM) added. Reverse transcriptase stops are marked by numbers starting from the A in the initiation AUG codon. The primer was hybridized 50 nt downstream of the AUG codon.

Figure 3

Figure 3

Footprints of tRNAs in the various functional complexes. Lane numbers correspond to (1) empty ribosomes; (2) ribosomes carrying MFK-mRNA and tRNAfMet; (3) ribosomes carrying MFK-mRNA, tRNAfMet and AcPhe-tRNAPhe; (4) ribosomes with MFK-mRNA, tRNAfMet, AcPhe-tRNAPhe and EF-G*GTP; (A, C, G, U) correspond to sequencing lanes, (K) to the unmodified rRNA. Modification with kethoxal was used in (A), CMCT in (B) and DMS in (C). Primers complementary to the 23S rRNA positions 2281–2301 (A), 2649–2667 (B) and 16S rRNA positions 1440–1458 (C) were used for the extension reactions. Primer extension stops corresponding to the nucleotides protected by tRNA are indicated, together with their tRNA binding site specificity.

Figure 4

Figure 4

Interaction of ribosomes carrying the C2394G mutation with EF-G, monitored by footprinting. (A) DMS footprints of EF-G on the GAC of the wild-type (WT) and mutant (C2394G) ribosomes. (A, C, G, U)—correspond to sequencing lanes; (K)—to the unmodified rRNA. Other lanes of the gel are marked according to the ribosomal complex, modified with DMS. (1) empty ribosomes; (2) ribosomes, MFK-mRNA, fMet-tRNAfMet, Phe-tRNAPhe*EF-Tu*GTP, EF-G*GMPPNP; (3) ribosomes, MFK-mRNA, fMet-tRNAfMet, Phe-tRNAPhe*EF-Tu*GTP, EF-G*GTP, fusidic acid; (4) ribosomes, EF-G*GMPPNP; (5) ribosomes, EF-G*GTP, fusidic acid. The primer extension stop corresponding to the nucleotide A1067 protected from DMS modification by EF-G is marked. (B) DMS footprints of EF-G on the Sarcin–Ricin loop of the wild-type and mutant ribosomes. Lanes are marked as in (A). The primer extension stop corresponding to the nucleotide A2660 protected from DMS modification by EF-G is marked.

Figure 5

Figure 5

Stimulation of the EF-G-catalysed GTPase reaction by complexes of wild-type ribosomes (WT), and ribosomes carrying the C2394G mutation (C2394G). (A) The time course of multiple turnover [γ-32P]GTP hydrolysis by EF-G, stimulated by either empty ribosomes or the complex of polyU-programmed ribosomes, carrying deacylated tRNAPhe in the P-site. (B) The dependences of the GTP hydrolysis rates on the concentration of EF-G. The hydrolysis was stimulated by either the empty ribosomes or the complex of polyU-programmed ribosomes with deacylated tRNAPhe. The curves are marked as indicated on the right-hand side of the graphs. Closed circles correspond to the polyU programmed wild-type ribosomes carrying deacylated tRNAPhe; grey circles correspond to the empty ribosomes; closed triangles correspond to the polyU programmed 2394G ribosomes carrying deacylated tRNAPhe; grey triangles correspond to the empty 2394G ribosomes.

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