How the ribosome moves along the mRNA during protein synthesis (original) (raw)
Related papers
Biochemistry, 1993
The ribosomal mRNA track was investigated by toeprinting 30s ribosomes, in the presence or absence of tRNA, using a variety of different ribosome-binding sites. We found that: (1) the ribosome, by itself, recognizes the mRNA translational initiation site; (2) the ribosomal mRNA track makes extensive contact with mRNA independent of tRNA and the start codon; (3) ribosomemRNA complexes are less stable than complexes containing tRNA; and (4) toeprinting can be used to analyze the contour of the ribosomal mRNA track, yielding information on its "height" as well as its "length" dimension. Examination of several ribosome-binding sites, including those containing very stable secondary structure, indicated that the "height" of the mRNA track is quite roomy, while the nucleotide distance between the site of Shine-Dalgarno annealing, the P site, and the 3'-edge of the mRNA track is fixed. The data suggest a mechanism for tethering regulatory elements to the ribosome during translation. Translational initiation is a multistep process which involves at least two RNA: RNA interactions: annealing of the 3'terminal nucleotides of the 16s rRNA to the Shine-Dalgarno (SD)' sequence and pairing of the initiator tRNA anticodon to the mRNA start codon (Calogero et al., 1988; Gualerzi et al., 1987). Initiation requires as well a variety of essential protein factors and mRNA regulatory elements that function during initiator tRNA selection and decoding of the start codon and contribute to the regulation of the level of translational expression [for recent reviews, see Rudd and Schneider (1992), Gualerzi and Pon (1990), McCarthy and Gualerzi (1 990), and Gold (1 988)]. Typically, the regulatory mRNA elements are contained within the ribosome-binding site (RBS), often comprising an unstructured region (Dreyfus, 1988) of about 35 nucleotides containing a purine-rich sequence 5-1 1 nucleotides upstream of the start codon (Storm0 et al., 1982). The regulatory effect of naturally occurring start codons, SD sequences, the spacing between them, and second codons on translational yield has been studied (Ringquist et al., 1992), as has the effect of stable secondary structure at the RBS (de Smit & Van Duin, 1990; Ringquist et al., 1992). Toeprinting, the inhibition of cDNA synthesis by the complex formed between mRNA, tRNA, and a ribosome, has provided an important technique for investigating the process of translational initiation (Hartz et al., 1989). A typical toeprint signal occurs 15 nucleotides 3' of the first nucleotide of the codon in the ribosomal P site and probably corresponds to the 3'-edge of the mRNA track (Hartz et al., 1988,1989; Kang & Cantor, 1985). Selection of the RBS by 30s particles (Hartz et al., 1989), decoding of initiator tRNA and the start codon by the interaction between protein initiation 7 This work was funded by a research grant from theNationa1 Institutes
Interactions of the ribosome with mRNA and tRNA
Current Opinion in Structural Biology, 2010
Recent collection of high-resolution crystal structures of the 70S ribosome with mRNA and tRNA substrates enhances our knowledge of protein synthesis principles. A novel network of interactions between the ribosome in the elongation state and mRNA downstream from the A codon suggests that mRNA is stabilized and aligned at the entrance to the decoding center. The X-ray studies clarify how natural modifications of tRNA are involved in the stabilization of the codon-anticodon interactions, prevention of frame-shifting and also expansion of the decoding capacity of tRNAs. In addition, the crystal structures provide the view that tRNA in the A and P sites communicate through a protein rich environment and suggest how these tRNAs are controlled through the intersubunit bridge formed by protein L31.
Nucleic Acids Research, 1992
Two tRNA molecules at the ribosomal A-and P-sites, with a relatively small angle between the planes of the L-shaped molecules, can be arranged in two mutually exclusive orientations. In one (the 'R'-configuration), the T-loop of the A-site tRNA faces the D-loop of the P-site tRNA, whereas in the other (the 'S'-configuration) the D-loop of the A-site tRNA faces the T-loop of the P-site tRNA. A number of stereochemical arguments, based on the crystal structure of 'free' tRNA, favour the R-configuration. In the ribosome, the CCA-ends of the tRNA molecules are 'fixed' at the base of the central protuberance (the peptidyl transferase centre) of the 50S subunit, and the anticodon loops lie in the neck region (the decoding site) of the 30S subunit. The translocation step is essentially a rotational movement of the tRNA from the Ato the P-site, and there is convincing evidence that the A-site must be located nearest to the L7/L12 protuberance of the 50S subunit. The mRNA in the two codon-anticodon duplexes lies on the 'inside' of the 'elbows' of the tRNA molecules (in both the S-type and R-type configurations), and runs up between the two molecules from the Ato the P-site in the 3' to 5'-direction. These considerations have the consequence that in the S-configuration the mRNA in the codon-anticodon duplexes is directed towards the 50S subunit, whereas in the R-configuration it is directed towards the 30S subunit. The results of site-directed cross-linking experiments, in particular cross-links to mRNA at positions within or very close to the codons interacting with Aor P-site tRNA, favour the latter situation. This conclusion is in direct contradiction to other current models for the arrangement of mRNA and tRNA on the ribosome.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1990
Using an RNA footprinting technique, accessible sites on the mRNA initiation region bound to the ribosome have been determined. Chemical probing experiments have been done both in the presence and absence of the initiator tRNA with dimethyl sulfate, kethoxal and carbodiimide as reagent probes. As an mRNA, a mini-mRNA containing the initiation region of bacteriophage ~ gene cro has been used. This region is characterized by a long single-stranded Shine-Dalgarno (SD) sequence followed by two hairpin structures of which the first one comprises in its loop the initiation codon. As compared to a free mRNA, the only nucleotides additionally protected in the binary mRNA-ribosome complex have been those which belong to the S-D sequence and the initiation codon.The protection of other nucleotides has not changed. Addition of the initiator RNA results in the modification of nuclcotides in the stems of the downstream hairpin structures of the initiation region. This reflects their transition into a single-stranded conformation promoted by tRNA. A possible implication of these findings for the decoding process is discussed.
Structural basis for messenger RNA movement on the ribosome
Nature, 2006
Translation initiation is a major determinant of the overall expression level of a gene 1-3 . The translation of functionally active protein requires the messenger RNA to be positioned on the ribosome such that the start/initiation codon will be read first and in the correct frame. Little is known about the molecular basis for the interaction of mRNA with the ribosome at different states of translation. Recent crystal structures of the ribosomal subunits 4-8 , the empty 70S ribosome 9 and the 70S ribosome containing functional ligands have provided information about the general organization of the ribosome and its functional centres. Here we compare the X-ray structures of eight ribosome complexes modelling the translation initiation, post-initiation and elongation states. In the initiation and post-initiation complexes, the presence of the Shine-Dalgarno (SD) duplex causes strong anchoring of the 59end of mRNA onto the platform of the 30S subunit, with numerous interactions between mRNA and the ribosome. Conversely, the 59 end of the 'elongator' mRNA lacking SD interactions is flexible, suggesting a different exit path for mRNA during elongation. After the initiation of translation, but while an SD interaction is still present, mRNA moves in the 39R59 direction with simultaneous clockwise rotation and lengthening of the SD duplex, bringing it into contact with ribosomal protein S2.
Contacts between the growing peptide chain and the 23S RNA in the 50S ribosomal subunit
Nucleic Acids Research, 1994
Peptides of defined length carrying a diazirine photoaffinity label attached either to the a-NH 2 group of the N-terminal methionine residue, or to the e-NH 2 group of an immediately adjacent lysine residue, were prepared in situ on Escherichia coli ribosomes in the presence of a synthetic mRNA analogue. Peptide growth was stopped simply by withholding the aminoacyl-tRNA cognate to an appropriate downstream codon. After photo-activation at 350 nm the sites of cross-linking to ribosomal RNA were determined by our standard procedures; the C-terminal amino acid of each peptide was labelled with tritium, in order to confirm whether the individual cross-linked complexes contained the expected 'full-length' peptide, as opposed to shorter products. The shortest peptides became cross-linked to sites within the 'peptidyl transferase ring' of the 23S RNA, namely to positions 2062, 2506, 2585 and 2609. However, already when the peptide was three or four residues long, a new crosslink was observed several hundred nucleotides away in another secondary structural domain; this site, at position 1781, lies within one of several RNA regions which have been implicated in other studies as being located close to the peptidyl transferase ring. Further application of this approach, combined with modelbuilding studies, should enable the path of the nascent peptide through the large ribosomal subunit to be definitively mapped. MATERIALS AND METHODS Preparation of mRNA, and tRNA derivatives An mRNA analogue was prepared by T7 transcription from a synthetic DNA template (9,11). This mRNA had the sequence GGG AGA AAG AAA AUG AAA UUC GAA CUG GAC ACC, carrying codons for methionine, lysine, phenylalanine and glutamic acid (M, K, F, E, underlined). Individual tRNA species
The EMBO journal, 1994
A photo-reactive diazirine derivative was attached to the 2-thiocytidine residue at position 32 of tRNA(Arg)I from Escherichia coli. This modified tRNA was bound under suitable conditions to the A, P or E site of E.coli ribosomes. After photo-activation of the diazirine label, the sites of cross-linking to 16S rRNA were identified by our standard procedures. Each of the three tRNA binding sites showed a characteristic pattern of cross-linking. From tRNA at the A site, a major cross-link was observed to position 1378 of the 16S RNA, and a minor one to position 936. From the P site, there were major cross-links to positions 693 and to 957 and/or 966, as well as a minor cross-link to position 1338. The E site bound tRNA showed major cross-links to position 693 (identical to that from the P site) and to positions 1376/1378 (similar, but not identical, to the cross-link observed from the A site). Immunological analysis of the concomitantly cross-linked ribosomal proteins indicated that S...