Multisite rate control analysis identifies ribosomal scanning as the sole high-capacity/low-flux-control step in mRNA translation.

Helena Firczuk,James Teahan,Pedro Mendes,John E.G Mccarthy

doi:10.1111/febs.15059

Helena Firczuk, James Teahan + Show 2 more

Open Access

https://doi.org/10.1111/febs.15059

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Abstract

Control of complex intracellular pathways such as protein synthesis is critical to organism survival, but is poorly understood. Translation of a reading frame in eukaryotic mRNA is preceded by a scanning process in which a subset of translation factors helps guide ribosomes to the start codon. Here, we perform comparative analysis of the control status of this scanning step that sits between recruitment of the small ribosomal subunit to the m7GpppG‐capped 5′end of mRNA and of the control exerted by downstream phases of polypeptide initiation, elongation and termination. We have utilized a detailed predictive model as guidance for designing quantitative experimental interrogation of control in the yeast translation initiation pathway. We have built a synthetic orthogonal copper‐responsive regulatory promoter (PCuR3) that is used here together with the tet07 regulatory system in a novel dual‐site in vivo rate control analysis strategy. Combining this two‐site strategy with calibrated mass spectrometry to determine translation factor abundance values, we have tested model‐based predictions of rate control properties of the in vivo system. We conclude from the results that the components of the translation machinery that promote scanning collectively function as a low‐flux‐control system with a capacity to transfer ribosomes into the core process of polypeptide production that exceeds the respective capacities of the steps of polypeptide initiation, elongation and termination. In contrast, the step immediately prior to scanning, that is, ribosome recruitment via the mRNA 5′ cap‐binding complex, is a high‐flux‐control step.

Highlights

Biological systems are generally highly complex and subject to multilayered control that can only be elucidated with the help of a combination of experimentation and computational modeling
A prime example of a complex biomolecular system is the protein synthesis machinery, which is responsible for creating all of the structures and functions that are associated with living cells [2,3,4]
Maintaining an efficient, high-precision mRNA translation machinery represents a major logistical and energetic burden for the cell, to the extent that, in the case of yeast, at least 76% of its total cellular energy budget is estimated to be committed to protein synthesis [5]

Summary

Introduction

Biological systems are generally highly complex and subject to multilayered control that can only be elucidated with the help of a combination of experimentation and computational modeling. Maintaining an efficient, high-precision mRNA translation machinery represents a major logistical and energetic burden for the cell, to the extent that, in the case of yeast, at least 76% of its total cellular energy budget is estimated to be committed to protein synthesis [5]. This machinery needs to be capable of accurate regulatory responses to environmental change [6]. At the heart of these key properties are features of control that are only beginning to be understood

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