Author(s): Akira Kaji
Protein synthesis has four steps, initiation, elongation of peptide chain, termination and the recycling of the spent ribosomes, mRNA and tRNA. The last step was discovered by our laboratories and catalyzed by a unique protein called ribosome recycling factor (RRF). The recycling step consists of three reactions, release of mRNA, tRNA and splitting of ribosomes. With the use of fluorescent labeled mRNA similar to the natural mRNA and labeled tRNA, we demonstrate in vitro that the major action of RRF is to release of mRNA and not the splitting of ribosomes into subunits. This corrects the general misconception that the major action of RRF is to split the ribosomes into subunits. The order of events with the naturally occurring substrate of RRF, the chain of events is release of tRNA, mRNA followed by the splitting of ribosomes. The release of mRNA is not dependent on the splitting of ribosomes. The in vitro results are supported by in vivo experiments where we used the translational coupling followed by the reporter gene expression (beta galactosidase expression). Using the basic reaction of RRF, release of ribosomes from mRNA, we developed a new screening system for the inhibitor of RRF. In this system, the inhibition of RRF reaction, makes ribosome stay on the mRNA at the termination codon, but start translating downstream which is linked to GFP. We show that this screening method functions by the use of known specific inhibitor of RRF, low concentration of fusidic acid. The assay method is simple and can be performed in 96 hole plate overnight. We look forward to finding collaborators who has access to the collection of possible inhibitors.
RNA phage GA coat and lysis protein articulation are translationally coupled through a covering end and commencement codon UAAUG. Basic for this coupling are the vicinity of the end codon of the upstream coat quality to the inception codon of the lysis quality (either a <3 nucleotide detachment or physical closeness through a potential barrette structure) yet not the Shine–Dalgarno arrangement. This recommends the ribosomes finishing the coat quality interpretation are solely liable for interpretation of the lysis quality. Inactivation of ribosome reusing factor (RRF), which ordinarily discharges ribosomes at the end codon, didn't impact the statement of the correspondent quality intertwined to the lysis quality. This recommends the likelihood that RRF may not discharge ribosomes from the intersection UAAUG. Be that as it may, RRF is basic for right ribosomal acknowledgment of the AUG codon as the commencement site for the lysis quality.
Catchphrases: commencement/reusing/ribosome/ribosome reusing factor (RRF)/translational coupling
Protein blend comprises of commencement, lengthening, end, and reusing of spent ribosomes for the following round of interpretation. The last advance is catalyzed by ribosome reusing factor (RRF) and prolongation factor G (EF-G). This progression dismantles the model post-end buildings (PoTCs, comprising of ribosomes, unesterified tRNA, and mRNA) into mRNA, tRNA, and ribosomes (for audit, see Kaji et al. 2001). The 70S ribosomes, the working type of ribosomes, must be separated into subunits for the commencement of protein amalgamation (Guthrie and Nomura 1968). In any case, the system of this critical advance isn't known.
Verifiably, this progression has been a wellspring of warmed conversation and has never been settled. One purpose of debate is whether the separation of ribosomes into subunits happens at the same time with the discharge from mRNA (Kaempfer 1970) or resulting to the arrival of 70S ribosomes from mRNA (Subramanian and Davis 1973; for an outline of this contest, see p. 154 of the survey by Janosi et al. 1996). Another purpose of debate is that Davis and his partners accepted that inception factor 3 (IF3) is a separation factor that separates 70S ribosomes into subunits (Subramanian and Davis 1970), while Kaempfer (1972) asserted that IF3 is an enemy of affiliation factor.
The specific job of inception factors in the subunit separation process has not been so clear either. In spite of the fact that IF3 and IF1 were accounted for to separate 70S ribosomes into subunits (Godefroy-Colburn et al. 1975), regardless of whether this is the genuine system by which ribosomes are separated into subunits is sketchy, in view of the accompanying reasons. The ribosome separation action of IF3 without anyone else can be watched uniquely under nonphysiological conditions, for example, a high grouping of IF3 or a low Mg2+ particle focus (Subramanian and Davis 1970; Kaempfer 1972). For sure, at 7 mM Mg2+ and without polyamines (and with 70 mM NH4+ and 30 mM K+), 3 μM IF3 and IF1 separate the 70S ribosome totally (Vila-Sanjurjo et al. 2004). Nonetheless, within the sight of polyamines (6 mM Mg2+, 2 mM spermidine, 0.05 mM spermine, and 60 mM NH4+), these two variables were not adequate to separate 70S ribosomes totally (Vila-Sanjurjo et al. 2004). We additionally saw that 4 μM IF3 (in vivo convergence of IF3 in developing Escherichia coli; Howe and Hershey 1983) doesn't separate 70S ribosomes under our traditional cushion conditions (8.2 mM Mg2+, no polyamines, and 80 mM NH4+) or in a support containing polyamines (5 mM Mg2+, 8 mM putrescine, 1 mM spermidine, 5 mM NH4+, 95 mM K+) which is enhanced for in vitro interpretation (Jelenc and Kurland 1979; Karimi et al. 1999). Besides, the conceivable job of IF1 in this procedure has been addressed (Kaempfer 1972).
Then again, a model has been proposed recommending that RRF, EF-G, and GTP separate 50S subunits from the PoTC, leaving 30S subunits and bound tRNA on mRNA (Karimi et al. 1999). This model doesn't clarify how mRNA is discharged from the PoTC. For additional subtleties of this model, see the Discussion area.
The information introduced beneath show that 70S ribosomes are briefly separated into subunits by RRF and EF-G, and that the subunits are then balanced out by IF3. Within the sight of IF3, the model PoTC is dismantled into stable subunits by EF-G and RRF. In this paper, we characterize "stable subunit separation" as "separation into subunits perceptible by sucrose thickness inclination centrifugation (SDGC)" and "transient subunit separation" as "separation into subunits discernible by the light dissipating decline however not by SDGC". At the point when we allude to just "subunit separation", this signifies "stable subunit separation". The steady subunit separation by the helpful activity of three components gives an answer for the longstanding crucial inquiry of how ribosomal subunits are accommodated the commencement venture of protein amalgamation.
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