Unusual ribosome binding properties of mRNA encoding bacteriophage λ repressor

Balakin, Andrey; Skripkin, Eugene; Shatsky, Ivan N.; Bøgdanov, A.

doi:10.1093/nar/20.3.563

Cited by 59 publications

(65 citation statements)

References 36 publications

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“…To analyze binding properties of leaderless mRNAs with the 40S and 80S ribosomes, we deliberately selected as a model an mRNA that starts with the 5Ј-terminal sequence of the leaderless phage cI mRNA, i.e., the same sequence which was used in similar studies with E. coli 70S ribosomes (1). The cIlacZ mRNA used in this work lacks all translation initiation regulatory elements inherent to eukaryotic or prokaryotic mRNAs and hence is an ideal model to reveal parallels between translation initiation on eukaryotic and prokaryotic ribosomes.…”

Section: Resultsmentioning

confidence: 99%

“…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.…”

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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: Resultsmentioning

confidence: 99%

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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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“…2B; anti-5′-UTR) reflects a structural peculiarity of the mRNA location on the 30S subunit: in a groove in isolated 30S subunits and a tunnel in 70S ribosomes (34,35). The inability of 70S ribosomes to initiate at internal initiation sites has been demonstrated (18,19). Thus, 30S subunits can initiate at any initiation site as long as the SD sequence is accessible, whereas 70S ribosomes must thread the mRNA from the 5′-end.…”

Section: Discussionmentioning

confidence: 99%

“…This initiation mode uses 70S ribosomes with the special feature that the ribosomal proteins S1 and S2 are not required, which are otherwise important for the 30S-binding mode (18). Initiation of lmRNA can even occur in the absence of all IFs (19,20). Additional information about lmRNAs is provided in SI Appendix, Introduction.…”

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70S-scanning initiation is a novel and frequent initiation mode of ribosomal translation in bacteria

Yamamoto

Wittek

Gupta

et al. 2016

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

106

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According to the standard model of bacterial translation initiation, the small ribosomal 30S subunit binds to the initiation site of an mRNA with the help of three initiation factors (IF1-IF3). Here, we describe a novel type of initiation termed "70S-scanning initiation," where the 70S ribosome does not necessarily dissociate after translation of a cistron, but rather scans to the initiation site of the downstream cistron. We detailed the mechanism of 70S-scanning initiation by designing unique monocistronic and polycistronic mRNAs harboring translation reporters, and by reconstituting systems to characterize each distinct mode of initiation. Results show that 70S scanning is triggered by fMet-tRNA and does not require energy; the Shine-Dalgarno sequence is an essential recognition element of the initiation site. IF1 and IF3 requirements for the various initiation modes were assessed by the formation of productive initiation complexes leading to synthesis of active proteins. IF3 is essential and IF1 is highly stimulating for the 70S-scanning mode. The task of IF1 appears to be the prevention of untimely interference by ternary aminoacyl (aa)-tRNA•elongation factor thermo unstable (EF-Tu)•GTP complexes. Evidence indicates that at least 50% of bacterial initiation events use the 70S-scanning mode, underscoring the relative importance of this translation initiation mechanism.protein synthesis | ribosomal functions | translational initiation | 30S-binding initiation | 70S-scanning initiation I t is textbook knowledge that 30S subunits initiate protein synthesis in bacteria; they recognize the initiation site of the mRNA composed of the Shine-Dalgarno (SD) sequence, the AUG codon, and fMet-tRNA, together with three initiation factors (IFs) forming the 30S initiation complex (30SIC). Association of the large 50S subunit triggers the release of the IFs, leading to the 70S initiation complex (70SIC) that enters the elongation phase of translation (reviewed in 1). We term this initiation path the "30S-binding mode" of bacterial initiation. After elongation and termination, it is thought that the ribosome dissociates into its subunits, thus providing 30S subunits for the next round of initiation.The functional role of IF2 is well defined. It can bind directly to the 30S, providing a docking site for fMet-tRNA (2), but it can also enter the 30S subunit as ternary complex fMet-tRNA•IF2•GTP (3). Both IF2 and IF3 are essential for viability. IF3 has a binding site at the 30S interface (4), which explains its antiassociation effect (5, 6), as well as its role in dissociation of the terminating 70S ribosome (7). However, the in vivo concentration of IF3 is 100-fold less (8) than required for full dissociation of 70S in vitro (4). Evidence for the presence of IF3 on 70S ribosomes was reported (9), indicating that the functional spectrum of IF3 is possibly not restricted to an antiassociation effect. Both IF3 and IF2 are also responsible for the fidelity of decoding the initiation AUG by fMet-tRNA Met f at the P site of 30S subun...

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“…A leaderless mRNA directly binds a 70S ribosome including an N-formyl-methionyl-transfer RNA, where translation is initiated (18)(19)(20). Leaderless mRNAs have been found in various species of prokaryotes, particularly in archaebacteria (21)(22)(23). For example, in Halobacterium salinarum, which belongs to the Euryarchaeota, leaderless mRNAs show a 15-fold higher activity in translation than mRNAs with the SD sequence (24).…”

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Dynamic evolution of translation initiation mechanisms in prokaryotes

Nakagawa

Niimura²,

Miura³

et al. 2010

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

128

167

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It is generally believed that prokaryotic translation is initiated by the interaction between the Shine-Dalgarno (SD) sequence in the 5′ UTR of an mRNA and the anti-SD sequence in the 3′ end of a 16S ribosomal RNA. However, there are two exceptional mechanisms, which do not require the SD sequence for translation initiation: one is mediated by a ribosomal protein S1 (RPS1) and the other used leaderless mRNA that lacks its 5′ UTR. To understand the evolutionary changes of the mechanisms of translation initiation, we examined how universal the SD sequence is as an effective initiator for translation among prokaryotes. We identified the SD sequence from 277 species (249 eubacteria and 28 archaebacteria). We also devised an SD index that is a proportion of SD-containing genes in which the differences of GC contents are taken into account. We found that the SD indices varied among prokaryotic species, but were similar within each phylum. Although the anti-SD sequence is conserved among species, loss of the SD sequence seems to have occurred multiple times, independently, in different phyla. For those phyla, RPS1-mediated or leaderless mRNA-used mechanisms of translation initiation are considered to be working to a greater extent. Moreover, we also found that some species, such as Cyanobacteria, may acquire new mechanisms of translation initiation. Our findings indicate that, although translation initiation is indispensable for all protein-coding genes in the genome of every species, its mechanisms have dynamically changed during evolution. dynamic evolution | Shine-Dalgarno sequence | ribosomal protein S1T ranslation initiation is fundamentally important for all protein-coding genes in the genome of every organism. Initiation, rather than elongation, is usually the rate-limiting step in translation, and proceeds at very different efficiencies depending on the sequences in the 5′ UTRs of mRNAs (1). In prokaryotes (for both eubacteria and archaebacteria), the Shine-Dalgarno (SD) sequence in an mRNA is well known as the initiator element of translation (2, 3). The SD sequence, typically GGAGG, is located approximately 10 nucleotides upstream of the initiator codon. The SD sequence pairs with a complementary sequence (CCUCC) in the 3′ end of a 16S rRNA. In the 16S rRNA, the sequence is called the anti-SD sequence in the 3′ tail of which region is single-stranded. The interaction between the SD and the anti-SD sequences (called the SD interaction) augments initiation by anchoring the small (30S) ribosomal subunit around the initiation codon to form a preinitiation complex (4). The importance of the SD interaction for efficient initiation of translation has been experimentally verified for both eubacteria and archaebacteria. Alterations of the SD sequence or the anti-SD sequence strongly inhibit protein synthesis, both in eubacteria including Escherichia coli (5) and Bacillus subtilis (6, 7) and in archaebacteria such as Methanocaldococcus jannaschii (8). For this reason, the SD interaction is thought to be the universal m...

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Unusual ribosome binding properties of mRNA encoding bacteriophage λ repressor

Cited by 59 publications

References 36 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

70S-scanning initiation is a novel and frequent initiation mode of ribosomal translation in bacteria

Dynamic evolution of translation initiation mechanisms in prokaryotes

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