proteins STRUCTURE O FUNCTION O BIOINFORMATICS Motion of transfer RNA from the A/T state into the A-site using docking and simulations Thomas Caulfield* and Batsal Devkota School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332 INTRODUCTION The ribosome is a large macromolecular complex com- prising protein and ribosomal RNA (rRNA). All cellular life requires ribosomes to synthesize proteins. The ribo- some decodes genetic information from messenger RNA (mRNA) with the assistance of transfer RNA (tRNA) and other cofactors. 1,2 Recent work has led to increased insights into ribosome function. 3–9 The composition of the ribosome is two-thirds rRNA and one-third protein. The sedimentation coefficient for an Escherichia coli ribo- some is 70S. The ribosome divides into two unequal halves, the large and small subunits (the LSU and SSU, respectively) [Fig. 1(B)]. The 70S ribosome has a mass of approximately 2.6 mega-Daltons, and the largest dimen- sion across the ribosome measures 220 A ˚ . The SSU is the decoding site, where mRNA and tRNA binding occurs to maintain the high fidelity of transla- tion. The literature suggests that its crystal structures indicate that nucleotides G530 and A1492/A1493 of 30S make contact at the minor groove of the mRNA and tRNA interaction. 10 The orientations of nucleotides G530 and A1492/1493 depend upon the presence of a cognate tRNA. 10 The ribosome assembles upon the mRNA to begin translation. This initiation begins with fMet-tRNA fMet . The fMet-tRNA fMet (initiator tRNA) binds at the P-site of the 30S subunit to form a preinitiation complex. Recruitment of the initiation factors (IF1, IF2, and IF3) causes the 50S subunit to form a complete 70S ribosome. Following this formation, the ternary complex aa*tR- NA*EF-Tu*GTP binds amnioacyl tRNA (aa*tRNA) to the T-site. The T-site is a transient site for the tRNA when Additional Supporting Information may be found in the online version of this article. Thomas Caulfield is currently affiliated at Department of Neuroscience, Mayo Clinic, Jacksonville, FL 32224, USA. Batsal Devkota is currently affiliated at Department of Bioinformatics, Reproduc- tive Medicine Associates of New Jersey, 111 Madison Ave, Morristown, NJ 07960, USA. *Correspondence to: Thomas Caulfield, Department of Neuroscience, Mayo Clinic, Jacksonville, FL 32224. E-mail: caulfield.thomas@mayo.edu or bdevkota@rmanj.com Received 23 January 2012; Revised 24 May 2012; Accepted 5 June 2012 Published online 22 June 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/prot.24131 ABSTRACT The ribosome catalyzes peptidyl transfer reactions at the growing nascent polypeptide chain. Here, we present a structural mechanism for selecting cognate over near-cognate A/T transfer RNA (tRNA). In part, the structural basis for the fidelity of translation relies on accommodation to filter cognate from near-cognate tRNAs. To examine the assembly of tRNAs within the ribonucleic–riboprotein complex, we conducted a series of all-atom molecular dynamics (MD) simulations of the entire solvated 70S Escherichia coli ribosome, along with its associated cofactors, proteins, and messenger RNA (mRNA). We meas- ured the motion of the A/T state of tRNA between initial binding and full accommodation. The mechanism of rejection was investigated. Using novel in-house algorithms, we determined trajectory pathways. Despite the large intersubunit cavity, the available space is limited by the presence of the tRNA, which is equally large. This article describes a ‘‘structural gate,’’ formed between helices 71 and 92 on the ribosomal large subunit, which restricts tRNA motion. The gate and the interacting protein, L14, of the 50S ribosome act as steric filters in two consecutive substeps during accommodation, each requiring: (1) sufficient energy contained in the hybrid tRNA kink and (2) sufficient energy in the Watson–Crick base pairing of the codon–anticodon. We show that these barriers act to filter out near-cognate tRNA and promote proofreading of the codon– anticodon. Since proofreading is essential for understanding the fidelity of translation, our model for the dynamics of this process has substantial biomedical implications. Proteins 2012; 00:000–000. V V C 2012 Wiley Periodicals, Inc. Key words: A/T transfer RNA; ribosome; docking; simulations; Maxwell’s demon molecular dynamics; structural fidelity. V V C 2012 WILEY PERIODICALS, INC. PROTEINS 1