Fidelity of translation in the presence of mammalian mitochondrial initiation factor 3

Ayyub, Shreya Ahana; Aswathy, S L; Dobriyal, Divya; Aluri, Srinivas; Spremulli, Linda L.; Varshney, Umesh

doi:10.1016/j.mito.2017.08.006

“…In this work, we investigated the effects of yeast mitochondrial IF3, Aim23p, on the Escherichia coli translation. The idea has come from the recent work of Ayyub et al (2018) where it has been demonstrated that mammalian mitochondrial IF3 (mtIF3), although being unable to fully substitute for IF3 in E. coli , exhibits some functional activity in bacterial cells. We exchanged the infC gene (coding for IF3) in bacteria by the AIM23 gene and found that Aim23p cannot substitute the cognate factor.…”

Section: Introductionmentioning

confidence: 99%

60S dynamic state of bacterial ribosome is fixed by yeast mitochondrial initiation factor 3

Левицкий

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Derbikova

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Baleva

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et al. 2018

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The processes of association and dissociation of ribosomal subunits are of great importance for the protein biosynthesis. The mechanistic details of these processes, however, are not well known. In bacteria, upon translation termination, the ribosome dissociates into subunits which is necessary for its further involvement into new initiation step. The dissociated state of the ribosome is maintained by initiation factor 3 (IF3) which binds to free small subunits and prevents their premature association with large subunits. In this work, we have exchanged IF3 in Escherichia coli cells by its ortholog from Saccharomyces cerevisiae mitochondria (Aim23p) and showed that yeast protein cannot functionally substitute the bacterial one and is even slightly toxic for bacterial cells. Our in vitro experiments have demonstrated that Aim23p does not split E. coli ribosomes into subunits. Instead, it fixes a state of ribosomes characterized by sedimentation coefficient about 60S which is not a stable structure but rather reflects a shift of dynamic equilibrium between associated and dissociated states of the ribosome. Mitochondria-specific terminal extensions of Aim23p are necessary for “60S state” formation, and molecular modeling results point out that these extensions might stabilize the position of the protein on the bacterial ribosome.

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“…Even if "60S state" is fixed in such conditions, it might rapidly dissociate to the subunits with the help of IF3. This may be also the case in the work of Ayyub and colleagues: having sufficient amounts of cognate E.coli IF3 allows bacterial ribosomes to keep the dissociated state in vivo properly regardless on the mammalian mtIF3 presence, and this could explain almost normal growth of the corresponding bacterial strains (Ayyub et al 2018). The question if "60S intermediate state" exists in wild-type bacterial cells is of high interest since it is worth proposing that in vitro ribosomes could not pose a conformation which is not pertained to them in vivo.…”

Section: Ribosome Purification and Analysismentioning

confidence: 83%

“…Interestingly, mammalian mtIF3 behaves quite differently in E.coli. Full-size factor does not markedly affect the E.coli growth rate while deletion of the N-terminal extension leads to the severe growth impairment (Ayyub et al 2018).…”

Section: Ribosome Purification and Analysismentioning

confidence: 94%

“…In this work, we investigated the effects of yeast mitochondrial IF3, Aim23p, on the E.coli translation. The idea comes from the recent work of Ayyub and colleagues where it has been demonstrated that mammalian mitochondrial IF3 (mtIF3), although being unable to fully substitute for IF3 in E.coli, exhibits some functional activity in bacterial cells (Ayyub et al 2018). We exchanged infC gene (coding for IF3) in bacteria by AIM23 gene and found that Aim23p cannot substitute the cognate factor.…”

Section: Introductionmentioning

confidence: 99%

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Supplemental Information 3: Raw Data for Fig2B

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The processes of association and dissociation of ribosomal subunits are of great importance for the protein biosynthesis. The mechanistic details of these processes, however, are not well known. In bacteria, upon translation termination, ribosome dissociates into subunits which is necessary for its further involvement into new initiation step. The dissociated state of ribosome is maintained by initiation factor 3 (IF3) which binds to free small subunits and prevents their premature association with the large subunits. In this work, we have exchanged IF3 in E.coli cells by its ortholog from Saccharomyces cerevisiae mitochondria (Aim23p) and showed that yeast protein cannot functionally substitute the bacterial one and is even slightly toxic for bacterial cells. Our in vitro experiments have demonstrated that Aim23p does not split E.coli ribosomes into subunits. Instead, it fixes an intermediate state of ribosomes dissociation characterized by sedimentation coefficient about 60S. Using molecular modeling, we show that such fixation is due to mitochondria-specific terminal extensions of Aim23p that stabilize the position of the protein on the bacterial ribosome.

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“…Full-length Aim23p is undesirable for E.coli cells due to its terminal extensions. 150 As it has been already mentioned in Introduction, mammalian mtIF3 possesses some 151 functional activity in E.coli cells (Ayyub et al 2018). On the other hand, we have previously 152 demonstrated that E.coli IF3 may partially rescue the growth defects of the yeast strain lacking 153 Aim23p (Kuzmenko et al 2014).…”

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confidence: 91%

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Supplemental Information 6: Initial Modeling of Aim23p Binding to 16S rRNA of E.coli

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The processes of association and dissociation of ribosomal subunits are of great importance for the protein biosynthesis. The mechanistic details of these processes, however, are not well known. In bacteria, upon translation termination, ribosome dissociates into subunits which is necessary for its further involvement into new initiation step. The dissociated state of ribosome is maintained by initiation factor 3 (IF3) which binds to free small subunits and prevents their premature association with the large subunits. In this work, we have exchanged IF3 in E.coli cells by its ortholog from Saccharomyces cerevisiae mitochondria (Aim23p) and showed that yeast protein cannot functionally substitute the bacterial one and is even slightly toxic for bacterial cells. 49 binds to the small subunit in order to keep it apart from the large one . This 50 stage is, in fact, the very first stage of the translation initiation process; 30S•IF3 complex 51 becomes the basis for the full-size initiatory complex formation which includes Shine-Dalgarno 52 sequence of mRNA, initiator tRNA, and initiation factors 1 and 2. It is worth mentioning that 53 anti-association activity of IF3 is definitely of passive mode: it does not promote dissociation of 54 the ribosome into subunits but instead binds to free small subunit and prevents its re-association 55 with the large one (Gualerzi et al. 1977) (Gottleib et al. 1975). 56The exact mechanism of ribosome dissociation into subunits remains not clear. This is 57 due to methodological complications of studying this fast and dynamic process. In kinetic study, 58 a model was proposed that assumed the existence of several consecutive conformations of 70S 59 ribosome in course of its dissociation; IF3 was hypothesized to be a potential effector of 60 corresponding conformational changes which could shift the equilibria between different states 61 of dissociating ribosome (Goss et al. 1980). It can be assumed that these conformations might be 62 characterized by different sedimentation coefficients, less than 70S but probably more than 50S.

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Fidelity of translation in the presence of mammalian mitochondrial initiation factor 3

Cited by 12 publications

References 37 publications

60S dynamic state of bacterial ribosome is fixed by yeast mitochondrial initiation factor 3

60S dynamic state of bacterial ribosome is fixed by yeast mitochondrial initiation factor 3

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