Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses

Abstract

Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis by antibiotics and during nascent polypeptide synthesis. Some of these antibiotics, and regulatory nascent polypeptides mostly in the form of peptidyl-tRNAs, inhibit either peptide bond formation or translation termination [1][2][3][4][5][6][7] . These inhibitory events can stop the movement of the ribosome, a phenomenon termed "translational arrest". Translation arrest induced by either an antibiotic or a nascent polypeptide has been shown to regulate the expression of genes involved in diverse cellular functions such as cell growth, antibiotic resistance, protein translocation and cell metabolism [8][9][10][11][12][13] . Knowledge of how antibiotics and regulatory nascent polypeptides alter ribosome function is essential if we are to understand the complete role of the ribosome in translation, in every organism.Here, we describe a simple methodology that can be used to purify, exclusively, for analysis, those ribosomes translating a specific mRNA and containing a specific peptidyl-tRNA 14 . This procedure is based on selective isolation of translating ribosomes bound to a biotin-labeled mRNA. These translational complexes are separated from other ribosomes in the same mixture, using streptavidin paramagnetic beads (SMB) and a magnetic field (MF). Biotin-labeled mRNAs are synthesized by run-off transcription assays using as templates PCR-generated DNA fragments that contain T7 transcriptional promoters. T7 RNA polymerase incorporates biotin-16-UMP from biotin-UTP; under our conditions approximately ten biotin-16-UMP molecules are incorporated in a 600 nt mRNA with a 25% UMP content. These biotin-labeled mRNAs are then isolated, and used in in vitro translation assays performed with release factor 2 (RF2)-depleted cell-free extracts obtained from Escherichia coli strains containing wild type or mutant ribosomes. Ribosomes translating the biotin-labeled mRNA sequences are stalled at the stop codon region, due to the absence of the RF2 protein, which normally accomplishes translation termination. Stalled ribosomes containing the newly synthesized peptidyl-tRNA are isolated and removed from the translation reactions using SMB and an MF. These beads only bind biotincontaining messages.The isolated, translational complexes, can be used to analyze the structural and functional features of wild type or mutant ribosomal components, or peptidyl-tRNA sequences, as well as determining ribosome interaction with antibiotics or other molecular factors 1,[14][15][16]

Protein Synthesis Factors (RF1, RF2, RF3, RRF, and tmRNA) and Peptidyl-tRNA Hydrolase Rescue Stalled Ribosomes at Sense Codons

“…[15][16][17][18][19][20][21][22][23][24][25] Non-programmed ribosomal stalling occurs in two different manners. First, it can occur with ribosome stalled at the end of non-stop mRNA.…”

Isolation of Ribosome Bound Nascent Polypeptides <em>in vitro</em> to Identify Translational Pause Sites Along mRNA

“…FLAG-peptide to ensure efficient pull down. An alternative protocol based on selective isolation of translating ribosomes bound to a biotin-labeled mRNA has been also recently decribed 13 . It should be noted that for more accurate determination of the translation pause sites, especially for large proteins one can use micrococcal nuclease protection assay 5 .…”