Flexibility of the nascent polypeptide chain within the ribosome

Abstract

The ribosomal environment of the N-terminus of the nascent polypeptide chain has been investigated using peptides of different lengths, synthesized in situ on Escherichia coli ribosomes ; the peptides each carry a photoreactive diazirine moiety at their N-terminus, so as to generate cross-links to neighbouring ribosomal components. Our previous studies [Choi, K. M. & Brimacombe, R. (1998) Nucleic Acids Res. 26, 887Ϫ895] with three independent families of peptides, derived from the E. coli ompA protein gene, the tetracycline-resistance gene and the bacteriophage T4 gene 60, identified a series of sites within the 23S rRNA to which the peptides became cross-linked. The distribution of these cross-links indicated that the nascent peptide is very flexible within the 50S subunit. Here, we demonstrate that the N-termini of the ompA and gene-60 peptides can, in addition, even become concomitantly cross-linked to the 30S subunit. The cross-linking is predominantly to 30S ribosomal proteins S1, S2, S4 and (to a lesser extent) S3, which form a cluster near to the decoding region. This result is discussed in terms of the flexibility of the nascent peptide during the co-translational folding process, and in terms of the 'ribosomal bypass' phenomenon which is known to occur during translation of the gene 60 mRNA.Keywords : protein synthesis; nascent peptide; photo-cross-linking; decoding site ; ribosomal bypass.During protein synthesis, individual amino acids are attached E. coli 50S ribosomal subunit by analysing cross-links to the 23S rRNA from a photoreactive reagent attached to the N-termito a nascent peptide chain by the peptidyl transferase reaction and, in due course, a completed polypeptide emerges from the nus of peptides of different lengths that were synthesized in situ on the ribosome [10Ϫ12]. The results of these studies, which large ribosomal subunit. However, the behaviour of the nascent peptide before its final emergence is a complex and controversial were with three independent families of peptides [12], showed that with increasing length, the peptides became progressively matter which has several interrelated aspects. First, there is the question of whether the path followed by the peptide is a tunnel cross-linked to sites within domains V, IV, II, III and I of the 23S rRNA. In particular, in each peptide family, the N-termini through the large subunit [1Ϫ3], or rather a channel along the subunit surface [4]. Secondly, there is the question of co-transla-of peptides of 30 amino acids or longer became cross-linked to a site on the 23S rRNA in the direct neighbourhood of sites that tional folding, that is to say the extent to which the peptide can already attain its final folded conformation within the ribosome had been previously cross-linked to L23 and L29 [13], proteins which, in turn, had been located by immune electron microscopy e.g. [5,6]. A third aspect is the regulatory influence of the na- [14] at positions close to the proposed exit site [9], mentioned scent peptide on the translation of its own ...

Alterations at the peptidyl transferase centre of the ribosome induced by the synergistic action of the streptogramins dalfopristin and quinupristin

“…But, once the P-site is occupied, binding of dalfopristin to the ribosome will be suppressed, thus explaining why ribosomes actively engaged in protein synthesis are not susceptible to S A (reviewed in [5]). The loss of flexibility of the CCA-end in the P-site concomitant with nascent chain extension [33] might enhance this effect, since the effectiveness of S A decreases as the number of amino acids attached to the P-site tRNA increases [30]. …”

Protein Folding by Domain V of Escherichia coli 23S rRNA: Specificity of RNA-Protein Interactions

“…The folding of globin (heme binding site formation) takes place on the PTC, and this intermediate is too large to go through the tunnel and has to leave via the interface between the two subunits (22). Some nascent peptide chains cross-link to 16S rRNA (10). The tail spike protein of phage P22 remains associated with the 30S subunit after the subunits dissociate (12).…”

Coupling of Open Reading Frames by Translational Bypassing

“…From this it seems reasonable to assume that the nascent peptide signal of gene 60 interacts with some feature of the large subunit of the ribosome. Yet, surprisingly, cross-linking studies with the nascent peptides from gene 60 and E. coli ompA indicate that both nascent peptides can interact with small subunit proteins in and around the decoding center, as well as around 23S rRNA and large subunit proteins (29,30). This unexpected conformational flexibility of the nascent peptide suggests the possibility that nascent peptides can directly alter tRNA-mRNA interactions on the 30S subunit.…”