Scanning model for translational reinitiation in eubacteria

Adhin, Malti R.; Duin, Jan van

doi:10.1016/s0022-2836(05)80265-7

Cited by 157 publications

(140 citation statements)

References 44 publications

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“…The 70S scanning also seems to work upstream in a few cases (in 7% of all IRs of E. coli, where 70S ribosomes after translation of a cistron should move upstream for limited distances of 1, 2, and 4 nt; SI Appendix, Fig. S1B), but occurs preferentially downstream as predicted from in vivo evidence (21).…”

Section: Discussionmentioning

confidence: 64%

See 1 more Smart Citation

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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Section: Discussionmentioning

confidence: 64%

“…a 70S type of bacterial initiation, has been conjectured several times previously (21)(22)(23), although no in-depth mechanistic evidence has verified this mode thus far. For example:…”

Section: Significancementioning

confidence: 99%

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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“…In the course of our earlier work on frameshifting (Falahee et al+, 1988;O'Connor et al+, 1989), we observed that several of our lacZ reporter gene constructs had Units of b-galactosidase obtained in wild-type or IF3 mutant strains carrying a lacZ plasmid with a UAG initiation codon (pSG500) and each of the indicated tRNA mutants on pRSVCATam1+2+5-derived-plasmids (Varshney & RajBhandary, 1990)+ Cells were grown in minimal medium with required supplements, tetracycline (12+5 mg/L) and ampicillin (200 mg/L)+ Each b-galactosidase activity measurement represents the mean (6 SD) of assays from at least three independent cultures+ unanticipated high levels of activity+ Subsequent mutagenesis and peptide sequencing of the expressed b-galactosidase showed that in many instances, such high basal levels of expression originated from initiation at internal sites within the mRNA, often at non-AUG codons+ Given the effects of the IF3 mutants on several atypical initiation events described above, we asked if mutations in IF3 affected such initiations from internal initiation codons+ Each lacZ frameshift construct contains an authentic AUG initiation codon and a downstream frameshifting window+ To synthesize b-galactosidase, (1) ribosomes must initiate from the authentic AUG codon and frameshifting must occur within this window delimited by stop codons or (2) initiation in a different reading frame must occur within the frameshifting window or downstream from it+ A variation on the latter possibility is that rather than de novo binding of 30S subunits to the internal start site, reinitiation at internal positions may occur by backscanning following termination at the downstream stop codon (Adhin & van Duin, 1990)+ In wild-type IF3 cells, the p304 ϩ1 frameshift construct displays a high basal ac-FIGURE 3. N-terminal sequencing to determine the influence of altered IF3 on initiation by mutant UAG-decoding initiator tRNA+ b-Galactosidase was purified from the R99L IF3 mutant containing the pSG500 plasmid and expressing the U29:A41 C30:G40 A31:U39/U35 A36 mutant initiator tRNA+ The yield in picomoles for selected PTH amino acids obtained for the first five cycles is shown+ The lower panel depicts the mRNA sequence and the experimentally determined amino acid sequence+ Units of b-galactosidase measured in wild-type or IF3 mutant strains carrying the indicated lacZ fusion plasmid+ Cells were grown in minimal medium with the required supplements and antibiotics+ Each number is the mean (6 SD) of assays from at least three independent cultures+ tivity (approximately 6% of wild-type lacZ ) and the presence of either IF3 mutation increases this activity fourfold (Table 1)+ Inspection of the p304 sequence (Table 4) shows the presence of a UUG codon in the ϩ1 reading frame 14 nt downstream of the AUG codon+ Alteration of this ϩ1 frame UUG codon in p304 to UUA (in plasmid p302) abolishes virtually all activity in wild-type and mutant IF3 strains, indicating that initiation from UUG is indeed responsible for the high expression seen in p304+ N-terminal sequencing of b-galactosidase isolated from the R99L IF3 mutant carrying p304 showed that initiation did indeed occur exclusively at the internal UUG codon in the IF3 mutant strain (Fig+ 4)+ Similarly, the p203 construct contains an internal GUG codon (Table 4) and has a high expression level in wild-type cells (387 units of b-galactosidase, Table 1)+ When this GUG codon in p203 was altered to CUG, CUA, AUC, or CUC, (constructs p222, p220, p211, and p212, respectively) expression diminished severely, indicating that initiation in p203 most likely derives from internal initiation at GUG+ When the act...…”

Section: If3 Modulates Initiation From Internal Initiation Sitesmentioning

confidence: 99%

Altered discrimination of start codons and initiator tRNAs by mutant initiation factor 3

O’Connor

Gregory²,

RajBhandary³

et al. 2001

RNA

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IF3 is essential for ensuring the fidelity of the initiation step of translation in bacterial cells. Mutations at residues R99 and R131 in the C-terminal domain of the factor have previously been shown to increase initiation from the noncanonical GUA codon. Here we show that these mutant forms of IF3 fail to discriminate against initiation from many different non-AUG codons. They also enhance the activity of mutant tRNAs carrying changes in the three consecutive G-C pairs that are conserved in the anticodon stem of initiator tRNAs. In addition, the IF3 mutants stimulate initiations from leaderless mRNAs and from internal initiation codons, in the absence of any SD-anti-SD interaction. These results indicate that IF3 ensures the accuracy of initiation by inspecting both the codon-anticodon pairing and unique features of the initiator tRNA as well as suppressing initiation from other potential start sites within the mRNA.

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“…The Tf start site can subsequently be reached by 'backscanning' over 8 nt (Adhin and Van Duin, 1990 (Fig. Id).…”

Section: Introductionmentioning

confidence: 99%

CCC.UGA: a new site of ribosomal frameshifting in Escherichia coli

Smit

Duin

Knippenberg

et al. 1994

Gene

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SUMMARYTo activate expression of a human transferrin(Tf)-encoding cDNA in Escherichia co/i by translational coupling, it was placed in an expression plasmid downstream from a S-terminal fragment from the replicase(R)-encoding gene of bacteriophage MS2. The resulting construct was found to produce, besides the desired Tf, a protein with the mobility of a fusion product (RTf) of the N-terminal R fragment and Tf. Analysis of available mutants showed that this fusion results from + 1 ribosomal frameshifting at the end of the R reading frame. This region contains the sequence, CCCUGA, suggesting that before termination occurs, tRNAP" may dislodge from the CCC codon and reassociate with the + 1 triplet CCU. By further site-directed mutagenesis, we demonstrate that both the CCC codon and the termination codon are indeed required for the observed 2-4% frameshifting. When either triplet is changed, the frequency of frameshifting drops to 0.3% or less. These results classify CCCUGA as a new '+ 1 shifty stop'.

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Scanning model for translational reinitiation in eubacteria

Cited by 157 publications

References 44 publications

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

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

Altered discrimination of start codons and initiator tRNAs by mutant initiation factor 3

CCC.UGA: a new site of ribosomal frameshifting in Escherichia coli

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