Specific bonding of puromycin to full-length protein at the C-terminus

Abstract

Puromycin, an analog of the 3' end of aminoacyl-tRNA, causes premature termination of translation by being linked non-specifically to growing polypeptide chains. Here we report the interesting phenomenon that puromycin acting as a non-inhibitor at very low concentration (e.g. 0.04 microM) can bond only to full-length protein at the C-terminus. This was proved by using a carboxypeptidase digestion assay of the products obtained by Escherichia coli cell-free translation of human tau 4 repeat (tau4R) mRNA in the presence of low concentrations of puromycin or its derivatives. The tau4R mRNA was modified to code for three C-terminal methionines, which were radioactively labeled, followed by a stop codon. The translation products could not be digested by carboxy-peptidase if puromycin or a derivative was present at the C-terminus of full-length tau4R. Puromycin and its derivatives at 0. 04-1.0 microM bonded to 7-21% of full-length tau4R, depending on the ability to act as acceptor substrates. Furthermore, the bonding efficiency of a puromycin derivative to tau4R was decreased by addition of release factors. These results suggest that puromycin and its derivatives at concentrations lower than those able to compete effectively with aminoacyl-tRNA can bond specifically to full-length protein at a stop codon. This specific bonding of puromycin to full-length protein should be useful for in vitro selection of proteins and for in vitro and in vivo C-terminal end protein labeling.

Puromycin oligonucleotides reveal steric restrictions for ribosome entry and multiple modes of translation inhibition

“…35 S]Met-puromycin (f-Met-puromycin) from a fragment of N-formyl-Met-tRNA f Met and puromycin (Monro & Marcker, 1967)+ Much of what we know about the catalytic activity of the ribosome has been derived from this simple assay (Noller et al+, 1992;Samaha et al+, 1995;Green & Noller, 1996)+ However, the reaction is conducted under conditions that are relatively nonphysiological (30% ethanol, 400 mM K ϩ , and 0-4 8C incubation temperature) and the effect of other critical components such as the template, small ribosomal subunit, and soluble translation factors cannot be measured, as they are absent+ Thus, the potency of puromycin may not be accurately reflected by its activity under fragment-reaction conditions+ To account for these shortcomings, some modifications have been made to more fully recapitulate the protein synthesis machinery+ Addition of the small subunit and poly(U)-mRNA followed by P-site charging with N-acetyl-Phe-tRNA Phe (Ac-Phe-tRNA Phe ) pushes the equilibrium towards subunit association, which allows peptidyl transferase to be carried out in the absence of alcohol (Chládek et al+, 1973)+ However, these adaptations still require salt-washed ribosomes, relatively high concentrations of NH 4 Cl and MgCl 2 to promote subunit assembly in the absence of initiation factors (Chládek et al+, 1973), and fail to yield long polypeptide-puromycin complexes (.12-15 amino acids in the Escherichia coli cell-free poly(A) system; Smith et al+, 1965)+ Thus, even these modifications result in systems that are biochemically removed from the complete translation apparatus+ To date, the majority of studies with translation inhibitors have been conducted using either the classical fragment reaction or a version of the f-MettRNA•A-U-G•70S or Ac-Phe-tRNA•poly(U)•70S system (Odom & Hardesty, 1992;Welch et al+, 1995;Michelinaki et al+, 1996;Green et al+, 1998)+ Interest in puromycin and puromycin conjugates has recently increased due to new methods for synthesizing molecular libraries (Nemoto et al+, 1997;Roberts & Szostak, 1997), photo-crosslinking reagents for the ribosome (Green et al+, 1998), peptidyl transferase inhibitors (Welch et al+, 1995;Nissen et al+, 2000), and in vitro synthesis of proteins containing specific labels or tags (Miyamoto-Sato et al+, 2000)+ Optimization of each of these processes requires that we understand how puromycin conjugates enter the ribosome in cis (when they are tethered to the mRNA in the decoding site) or in trans when these molecules enter the ribosome from solution+ Considerable effort has recently gone into understanding the cis reaction to facilitate the synthesis of the mRNA-peptide fusions for mRNA-display selection experiments (Liu et al+, 2000)+ Interestingly, the trans reaction between puromycin con...…”

Cell-free cotranslation and selection using in vitro virus for high-throughput analysis of protein–protein interactions and complexes

“…To further decrease false positives and to obtain information about direct/indirect interactions, an in vitro postselection was performed. The post-selection is composed of a pull-down assay to confirm the interactions using C-terminal protein labeling (Nemoto et al 1999;Miyamoto-Sato et al 2000Doi et al 2002) and real-time polymerase chain reaction (PCR) assay to confirm the enrichments. The use of two-step purification of the IVV selection followed by post-selection should provide reliable data for PPI analysis.…”

Development of a microscopic platform for real-time monitoring of biomolecular interactions

“…In the same way, the preparation of fluorescently labeled target proteins also becomes straightforward as demonstrated in this study (Figs. 5,6), by using in vitro protein synthesis-coupled fluorescent labeling (Nemoto et al 1999;Miyamoto-Sato et al 2000;Doi et al 2002).…”