Conversion of 48S translation preinitiation complexes into 80S initiation complexes as revealed by toeprinting

Dmitriev, Sergey E.; Pisarev, Andrey V.; Rubtsova, Maria P.; Dunaevsky, Yan E.; Shatsky, Ivan N.

doi:10.1016/s0014-5793(02)03776-6

Cited by 64 publications

(92 citation statements)

References 25 publications

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“…The reconstitution of 48S complexes from purified components and their analysis by toeprinting were performed as described previously (5,6,16,17). For the assembly and toeprinting analysis of 80S translation initiation complexes, the reaction mixture (20 l) contained 80S reassociated ribosomes (2.5 pmol), an mRNA (0.5 pmol), MettRNA i Met (5 pmol), and, where indicated, the initiation factors at the same molar ratio as for the assembly and analysis of 48S translation initiation complexes (5).…”

Section: Methodsmentioning

confidence: 99%

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A Leaderless mRNA Can Bind to Mammalian 80S Ribosomes and Direct Polypeptide Synthesis in the Absence of Translation Initiation Factors

Andreev

Terenin

Dunaevsky

et al. 2006

Molecular and Cellular Biology

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Translation initiation in eukaryotic cells is known to be a complex multistep process which involves numerous protein factors. Here we demonstrate that leaderless mRNAs with initiator Met-tRNA can bind directly to 80S mammalian ribosomes in the absence of initiation factors and that the complexes thus formed are fully competent for the subsequent steps of polypeptide synthesis. We show that the canonical 48S pathway of eukaryotic translation initiation has no obvious advantage over the 80S pathway of translation initiation on leaderless mRNAs and suggest that, in the presence of competing mRNAs containing a leader, the latter mechanism will be preferred. The direct binding of the leaderless mRNA to the 80S ribosome was precluded when such an mRNA was supplied with a 5 leader, irrespective of whether it was in a totally single-stranded conformation or was prone to base pairing. The striking similarity between the mechanisms of binding of leaderless mRNAs with mammalian 80S or bacterial 70S ribosomes gives support to the idea that the alternative mode of translation initiation used by leaderless mRNAs represents a relic from early steps in the evolution of the translation apparatus.More than a decade ago, we showed using the toeprint assay that the prokaryotic leaderless mRNA coding for the cI repressor of lambda phage assisted by initiator Met-tRNA was able to bind directly to undissociated 70S ribosomes in the absence of initiation factor 1 (IF1), IF2, and IF3 (1). Under the same conditions, no initiation complex was observed with the 30S ribosomal subunit. In the presence of initiation factors, the 30S subunit invariably bound to internal sites on the mRNA immediately downstream of the closest Shine-Dalgarno (SD) sequence, whereas undissociated 70S ribosome strongly preferred the initiation triplet as close as possible to the 5Ј end of the mRNA. These studies were substantially extended and confirmed in other laboratories both in vitro and in vivo (12,13,15,28) and resulted in similar conclusions. Natural leaderless mRNAs have been identified in all kingdoms of life (see references 10 and 11). This class of mRNAs is well represented in archaea and mammalian mitochondria. Although they have not yet been found among cellular mammalian mRNAs, leaderless monocistronic N and dicistronic N-P RNAs of measles virus were found to be associated with polysomes in infected mammalian cells (4). In addition, it is unlikely that a systematic search for such mRNAs in mammalian cells has ever been undertaken. There is one well-documented example of a translatable leaderless mRNA in unicellular eukaryotes. It encodes a variant specific surface protein of Giardia lamblia and is certainly translated in these cells (14). Leaderless mRNAs are efficiently translated in mammalian cell-free systems (see reference 12). However, it is not known whether they use a similar (80S) pathway for translation initiation in eukaryotes, given the remarkable quantitative and qualitative differences in the organization of eukaryotic and prokaryotic t...

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Section: Methodsmentioning

confidence: 99%

“…Primer extension analysis was performed with the oligonucleotide 5Ј-CCAGGGTTTTCCCAGTCACG-3Ј that is complementary to nucleotides 63 to 83 from the 5Ј end of cIlacZ mRNA. Analysis of the resulting cDNA was performed using denaturing 6% PAGE as described before (5). Radioactive bands were visualized with a PhosphorImager (Molecular Dynamics).…”

Section: Methodsmentioning

confidence: 99%

A Leaderless mRNA Can Bind to Mammalian 80S Ribosomes and Direct Polypeptide Synthesis in the Absence of Translation Initiation Factors

Andreev

Terenin

Dunaevsky

et al. 2006

Molecular and Cellular Biology

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“…Furthermore, the distance from the first nucleotide of the codon-like trinucleotide of PKI (cytosine of the CCU in the TSV IRES, numbered +1) to the mRNA entrance is 12-13 nt, as the toe prints of +13 to +14 report (7,10,17). It is known from the studies on bacterial 70S • tRNA initiation complexes (46,47), as well as on mammalian 80S ribosomes (48) bound with initiator methionyl-tRNA Met and/or IRESs initiating with the canonical AUG codon, including the hepatitis C virus (49) and encephalomyocarditis virus (50) IRESs, that the toe prints of +16 to +17 correspond to the placement of the AUG codon in the P site. The toe prints of +13 to +14, observed for the PSIV, CrPV, and TSV IRESs on eukaryotic ribosomes (7,10,17), are therefore consistent with the placement of the CCU trinucleotide of PKI in the A site in both the 40S and 80S complexes.…”

Section: Tsv Ires Stabilizes Two Partially Rotated Conformations Of Thementioning

confidence: 99%

Taura syndrome virus IRES initiates translation by binding its tRNA-mRNA–like structural element in the ribosomal decoding center

Koh

Brilot

Grigorieff

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

116

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In cap-dependent translation initiation, the open reading frame (ORF) of mRNA is established by the placement of the AUG start codon and initiator tRNA in the ribosomal peptidyl (P) site. Internal ribosome entry sites (IRESs) promote translation of mRNAs in a capindependent manner. We report two structures of the ribosomebound Taura syndrome virus (TSV) IRES belonging to the family of Dicistroviridae intergenic IRESs. Intersubunit rotational states differ in these structures, suggesting that ribosome dynamics play a role in IRES translocation. Pseudoknot I of the IRES occupies the ribosomal decoding center at the aminoacyl (A) site in a manner resembling that of the tRNA anticodon-mRNA codon. The structures reveal that the TSV IRES initiates translation by a previously unseen mechanism, which is conceptually distinct from initiator tRNA-dependent mechanisms. Specifically, the ORF of the IRES-driven mRNA is established by the placement of the preceding tRNA-mRNA-like structure in the A site, whereas the 40S P site remains unoccupied during this initial step.tRNA-mRNA mimicry | IRES-dependent initiation | factor-independent initiation | FREALIGN | real-space refinement

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“…4a [lanes 3,4,[7][8][9][10][11][12][13][14][15][16] and 4b [lanes 5-8]). However, alterations in termination factor activity yielded different toeprint phenotypes.…”

Section: Yeast; Mrna Circularization; Translation Initiation; Terminamentioning

confidence: 99%

Translation factors promote the formation of two states of the closed-loop mRNP

Amrani

Ghosh

Mangus

et al. 2008

Nature

179

157

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Efficient translation initiation and optimal stability of most eukaryotic mRNAs depends on the formation of a closed loop structure and the resulting synergistic interplay between the 5′ m 7 G cap and the 3′ poly(A) tail 1,2 . Evidence of eIF4G and Pab1p interaction supports the notion of a closed loop mRNP 3 , but the mechanistic events that lead to its formation and maintenance are still unknown. Here we have used toeprinting and polysome profiling assays to delineate ribosome positioning at initiator AUG codons and ribosome:mRNA association, respectively, and find that two distinct stable (cap analog resistant) closed loop structures are formed during initiation in yeast cell-free extracts. The integrity of both forms requires the mRNA cap and poly(A) tail, as well as eIF4E, eIF4G, Pab1p, and eIF3, and is dependent on the length of both the mRNA and the poly(A) tail. Formation of the first structure requires the 48S ribosomal complex whereas the second requires an 80S ribosome and the termination factors eRF3/Sup35p and eRF1/Sup45p. Surprisingly, the involvement of the termination factors is independent of a termination event. Keywords Yeast; mRNA circularization; translation initiation; termination factorsIn vitro translation reactions utilized synthetic mRNAs derived from yeast transcripts, extracts that recapitulate cap/poly(A) tail synergy ( Supplementary Fig. 1a), competitive inhibition of translation initiation by m 7 GpppG (cap analog), and analyses of ribosome positioning or mRNA association by toeprinting and sucrose gradient sedimentation. Addition of the elongation inhibitor, cycloheximide (CHX), to translation reactions programmed by the 2135nt AAA and UAA mRNAs containing long or short ORFs, respectively, (Fig. 1a) allowed detection of CHX-dependent initiator AUG toeprints that reflect 80S ribosomes protecting 16-18 nt 3′ of the AUG 4-6 (Fig. 1b, top and middle panels, lanes 1 and 3). These toeprints were dependent on initiation codon recognition, the presence of yeast extract, and concurrent mRNA translation ( Supplementary Fig. 1b and c). Supporting the latter conclusion, toeprints were almost completely eliminated by 2.7mM cap analog, a concentration that distinguished bona fide toeprints from background bands (Fig. 1b, top and middle panels, lanes 2 and 4). Lower cap analog concentrations also inhibited AUG toeprint accumulation, with 70% and 96% sensitivity obtained at 0.05mM and 0.5mM, respectively (Fig. 1b, lower panel). A shorter mRNA (miniUAA1, 488nt, Fig. 1a) also yielded the AUG toeprint (Fig. 1c,upper panel, lane 1), but this band was resistant to 2.7mM cap analog (lane 2) and only manifested sensitivity at higher concentrations (Fig. 1c, lower panel). Thus, in wild-type extracts, the short capped (see Supplementary Fig. 1d) and polyadenylated miniUAA1 mRNA is ∼160-fold more resistant to cap analog than the longer AAA mRNA. The miniADE2 (485nt) and ADE2 (2070nt) mRNAs, whose respective sizes (but not sequences) are comparable to those of the miniUAA1 and AAA transcripts, also...

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Conversion of 48S translation preinitiation complexes into 80S initiation complexes as revealed by toeprinting

Cited by 64 publications

References 25 publications

A Leaderless mRNA Can Bind to Mammalian 80S Ribosomes and Direct Polypeptide Synthesis in the Absence of Translation Initiation Factors

A Leaderless mRNA Can Bind to Mammalian 80S Ribosomes and Direct Polypeptide Synthesis in the Absence of Translation Initiation Factors

Taura syndrome virus IRES initiates translation by binding its tRNA-mRNA–like structural element in the ribosomal decoding center

Translation factors promote the formation of two states of the closed-loop mRNP

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