Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Summary

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM “arrest sequence” can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Abstract

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process^1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.

SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon⁶. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site^7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes⁷. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo de E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.

The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains^10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

Protocol

1. DNA Template Preparation and in vitro Transcription The gene of interest is cloned in any T7 and/or e.g. SP6 promoter based plasmid. To obtain RNCs of interest, C-terminus of the target polypeptide is extended by adding arrest inducing sequence of SecM FXXXXWIXXXXGIRAGP7. In order to ensure that the polypeptide fragment of interest will extrude out of the ribosomal tunnel, the C-terminal part of the target protein has to be extended by at least 30 amino acids12-14. A flexible…

Discussion

For reproducible results, quality and concentration of the components used for in vitro transcription and translation are critical. We have used commercially available in vitro transcription and translation extracts and they give efficient and reproducible results, if handled carefully. Quality of mRNA can affect the translation, so it is of utmost importance to test the integrity of mRNA before using it for in vitro translation. Incubation time for in vitro translation varies with the…

Materials

Name of reagent/ Kit	Company	Catalogue number
MEGAscript T7 High yield Transcription Kit	Ambion	AM1333
RTS 100 E.coli HY Kit	5 Prime	2401100
Ribonuclease Inhibitor	Invitrogen	15518012
Trans [35S]-Label	MP Biomedicals	0151006
Amicon Ultra-4 Centrifugal Filter Unit	Millipore	UFC801008
Storage phosphor autoradiography	GE Healthcare	Typhoon 9410 variable mode imager
Density Gradient Fractionation Systems	Teledyne Isco, Inc.	ISCO Programmable Density Gradient System
Sucrose Gradient Centrifugation	Beckman Coulter	Optima L-90 K Preparative Ultracentrifuge