Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

Summary

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Abstract

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the message^{for review see 1}. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates¹. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others². Codon bias is organism as well as tissue specific^2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs⁴. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes^5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein folding^{for review see 9}. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding^1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.

Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems^6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Protocol

1. DNA Template Preparation and in vitro Transcription

1. The gene of interest is cloned under T7 and/or e.g. SP6 transcriptional promoter.
2. For in vitro transcription the template DNA is linearized with an appropriate restriction enzyme cutting downstream of the ORF stop codon and/or mRNA 3' end. One needs to verify complete linearization of the plasmid DNA by running the restriction digestion product on agarose gel electrophoresis.
3. The linearized plasmid is used for in vitro transcription reaction. Different concentration of template DNA can be tested to identify optimum DNA concentration required for in vit....

Discussion

For reproducible results, quality and concentration of the components used for in vitro transcription and translation reactions are critical. In the current study we have used commercially available kits and extracts that provide highly reproducible data, if handled carefully. However, translation-competent extracts can be prepared from the cell of one's choice, if needed. Quality of mRNA can affect the translation, so it is of utmost importance to test the integrity of mRNAs before using it during in vitro<.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
MEGAscript T7 High yield Transcription Kit	Ambion	AM1333
Ribonuclease Inhibitor	Invitrogen	15518012
Trans [35S]-Label	MP Biomedicals	0151006
Ribonuclease-A	Invitrogen	12091
Rabbit Reticulocyte Lysate System, Nuclease Treated	Promega	L4960
E. coli S30 Extract System for Linear Templates	Promega	L1030
Centrifugation	Beckman Coulter	Optima TLX Ultracentrifuge
Storage phosphor autoradiography	GE Healthcare	Typhoon 9410 variable mode imager
Software for nascent polypeptide analysis	GE Healthcare	Image Quant TL, v2005