54NISR Research GRANT
2007 Young Investigator Research Grant

Comprehensive analysis of the effect of E. coli proteins on thetranslation reaction and its application to protein production
Tomoaki MATSUURA
Department of Bioinformatic Engineering, Osaka University

Research aims

Results

The protein translation reaction, one of the most important regulators of cell behavior, involves the interactions ofa large number of components, and can thus be seen as anintermolecular interaction network. It has been demonstrated experimentally that 36 proteins and the ribosomes
are sufficient to carry out protein translation.1 These minimal protein components include the ribosomal proteins, initiation, elongation, and release factors, aminoacyl-tRNA
synthetases, and enzymes involved in energy regeneration.
While the genome of Escherichia coli contains more than
4000 genes,2 constituting a very large interaction network,3
the number of protein components constituting the protein
synthesis system corresponds to only 2.1% of the genes encoded in the genome. Thus, only small subsets of the protein components are required for protein synthesis. On theother hand, a number of previous studies, including protein–protein interaction (PPI) network analysis in E. coli,3 indicated that protein components constituting the minimal protein synthesis system interact with a large number of otherproteins. To gain a deeper understanding of the proteintranslation system, it is important to identify not only theproteins that interact physically but also those that interact
functionally, i.e., those that affect the activity of the translation machinery. While there have been many studies tocharacterize the properties of such individual proteins indetail, there have been no previous attempts to search forsuch proteins in the entire genome. Hence, it is not clear
what fraction of gene products of E. coli are linked functionally with the minimal protein synthesis system.

By measuring the effects of individual ORF products on
the GFP synthesis reaction using the PURE system, we
demonstrated that at least 12% of the 4194 ORF productsof E. coli can affect the activity of the system (Fig. 1).4 We
designated these as functional modifiers of the protein synthesis reaction composed of minimal protein components.
We found 10 ORF products that completely suppressed
the GFP synthesis reaction, in addition to many that decreased the efficiency to a lesser extent. These genes included those encoding the transcriptional repressor LacI(GFP DNA construct used contained the lacO region), toxins (ChpA, ChpB, RelE, and YoeB) known to exhibit ribosome-dependent nuclease activity, and the nuclease Rnt.
Thus, our data were consistent with previous observations.
On the other hand, functionally uncharacterized proteins
were also found to be lethal for the reaction. To verify thatthe beneficial components are effective when added to thereaction as purified proteins, six proteins were overexpressed and purified from E. coli. While the optimum concentration for each protein differed, addition of six purified

Methods
In the present study, we performed an experimental searchfor open reading frame (ORF) products of the E. coligenome that affect the activity of the translation system. The
PURE system is an E. coli-based protein synthesis systemcomposed of the minimal protein components1. As this is areconstituted system comprised of defined components, it
differs from other conventional cell extract-based in vitrotranslation systems, which contain a number of unidentified
components. We measured the effects of 4194 individual
ORF products on the green fluorescent protein (GFP) synthesis reaction using the PURE system.

Fig. 1. The effect of E. coli ORF products on the GFP synthesis reaction
Vertical axis shows the GFP fluorescence intensity obtained after 3 h in thepresence of each ORF products during the GFP synthesis reaction. Numbers on the horizontal axis indicate the ranking of each ORF gene assigned
according to its fluorescence intensity among the 4194 ORFs and 62 independent measurements of the ybaW gene. Number 1 is the ORF that gavethe lowest value.

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proteins resulted in an increase in the fluorescence intensity.
Furthermore, when all six proteins (HrpA, Orn, PhnH,SlyD, Tig, and TrxC) were added at the optimum concentrations, the fluorescence intensity increased by 2.44-fold.
The PPI network shows the physical interactions between
the proteins, and such networks from various organisms
have been investigated; the results of these studies have indicated that proteins are highly connected with each other.
As the protein translation system is embedded in such alarge interaction network, we were also interested in thetopological relationships between the minimal components
and those that are functionally linked with them in the PPI
network of E. coli, which may provide insight into the topological structure and evolution of the protein synthesis system. We thus mapped each of the components involved in
the protein synthesis reaction on the PPI network of E.
coli.3 Network analyses indicated that functional modifiers
seem to be spread across the PPI network rather than clustering close to the minimal protein components.4 A possible
interpretation of this observation in relation to the evolutionary process of the protein synthesis system is that thesystem may have evolved by first establishing a system withminimal components, and then adding components further
outside rather than integrating them into the network composed of the minimal components.

Conclusion
The protein translation reaction, one of the most important regulators of cell behavior, can be operated by 91 protein components (55 ribosomal proteins36 factors), representing only 2.1% of the entire complement of ORFs in E.
coli. Through comprehensive analysis, we showed experimentally that at least 12% of the E. coli genes affected theactivity of the minimal protein synthesis system. The comprehensive data obtained in this study will be useful in future studies on individual proteins as well as functional genomics and systems biology. While we are still at the starting point of investigating the precise roles of the modifierproteins on the reaction, combining the experimental datawith the PPI network may provide insight into how these
proteins interact with the translation machinery. Moreover,
our data may be useful not only for technologies utilizing invitro translation systems, but also for designing in vitrotranslation systems with significantly improved performance than those available at present and also and for protein
production in vivo, such as by coexpressing beneficial components together with a protein of interest.
References
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Shimizu, Y. et al. Cell-free translation reconstituted with purifiedcomponents. Nat. Biotechnol. 19, 751–755 (2001).
Riley, M. et al. Escherichia coli K-12: a cooperatively developed annotation snapshot—2005. Nucleic Acids Res. 34, 1–9 (2006).
Butland, G. et al. Interaction network containing conserved and essential protein complexes in Escherichia coli. Nature 433, 531–537(2005).
Kazuta, Y. et al. Comprehensive analysis of the effects of Escherichia
coli ORFs on protein translation reaction. Mol Cell Proteomics 7,1530–1540 (2008).