Biophysics

Choreographed movement – unlocking the inner workings of the in protein synthesis

Ribosomes, tiny organelles within our cells, conduct the highly sequence for a particular protein will list the choreographed movements of protein synthesis. They ensure that the codons in the order in which the respective amino acids must be linked to one another. information encoded in DNA is successfully translated into proteins However, a ribosome cannot process the essential for nearly all chemical processes in our body, with an error rate string of codons while it is contained within of just 1 in 5000. Dr Karissa Sanbonmatsu and her team at Los Alamos DNA. The DNA must be unwound and National Laboratory, New Mexico, use cutting edge computational and copied onto a single strand called mRNA (messenger RNA). imaging tools to investigate exactly how these extraordinary structures work. Specifically, they focus on translocation, one of the most complex are made up of two subunits (see and little understood processes in protein synthesis. Figure 1): a small subunit where the mRNA is attached for decoding of the genetic instructions and a large subunit where the ibosomes are only 25 nanometers order specified. In other words, ribosomes respective amino acids are added to form (billionths of a meter) in diameter. convert genetic language into the language the growing protein. Despite this, they are one of the of proteins. most essential cellular organelles The mRNA strand attaches to the small involved in protein synthesis. But how does the ribosome decode the subunit of the ribosome which reads They provide the scaffold upon which the cell’s genetic information? Proteins are through each codon one at a time. Another Rdecoding of the cell’s genetic material is made up of small amino acids and how many molecule, transfer RNA (tRNA), acts as the realised, taking a string of RNA (think of amino acids are used, and in which order, is crucial intermediary. It has a single sequence it as a recipe book or instruction manual) specific to each protein. Each amino acid is of three base letters, an ‘anticodon’, and and building a protein from amino acids coded for in DNA by a ‘codon’ of three base carries the corresponding amino acid that (the ingredients or building blocks) in the letters e.g. AUG or CCA or UGA. A DNA is encoded by the complementary codon on the mRNA. Think of a lock and a key, where the lock is the mRNA codon and the key is the tRNA anticodon. As soon as the Figure 1: The two tRNA approaches the small subunit of the Ribosome Amino acid subunits of the ribosomes ribosome, it tries to fit on to the codon that is currently exposed on the mRNA strand. Large If the anticodon on the tRNA matches the subunit Amino acid codon on the mRNA, then the fit is strong and the tRNA will be accepted into the ribosome (accommodation). The amino acid carried by the tRNA will bind to the previous one in the sequence and the growth of the protein – on the large subunit – commences.

U A C G G U A MODEL EXPLANATION

A U G C C A It sounds relatively simple, but for the past Small U G mRNA AA A 40 years researchers have focused on the subunit movement of tRNA through the ribosome Large-scale computer simulation of the ribosome using supercomputers at Los Alamos National Laboratory. without offering any adequate or logical Newton's equations of motion are solved several million explanation for the underlying molecular times for each atom in the ribosome and each atom in the surrounding solution environment (total of 2.6 START codon Read head mechanism that dictates this process. In million atoms). White, large subunit RNA; magenta, fact, there are many cases where the tRNA small subunit RNA; light green, large subunit proteins; fit is weak, indicating that it is carrying the pink, small subunit proteins; yellow, aminoacyl tRNA; cyan, peptidyl tRNA; green ribbon, messenger RNA; Decoding wrong amino acid sequence. When this green spheres and red spheres, amino acids. happens, the tRNA is physically rejected by

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the ribosome. This is where the work of Dr Karissa Sanbonmatsu demonstrates its Detail profound significance and brilliance. Dr Sanbonmatsu is a principal investigator RESEARCH OBJECTIVES at Los Alamos National Laboratory, New Ribosomes provide Dr Sanbonmatsu’s research is focused Mexico – one of the most famous national Can you briefly explain to us the the scaffold upon on using molecular simulations to institutions in the US. She has dedicated principles of the computer model you understand tRNA movement through her research to using computational and have developed for understanding the which the decoding the ribosome and interpret cryo-EM experimental approaches to understand molecular mechanism behind tRNA of the cell’s genetic data. She is also using chemical probing the mechanism of a diverse array of non- translocation? to study structures of long non-coding coding RNA systems, including ribosomes, Our model focuses on data from single material is realised in her wet lab. riboswitches and long non-coding RNAs. molecule fluorescence experiments and cryo electron microscopy (cryo- specific step that the antibiotic blocks, FUNDING Her current focus aims to take advantage EM). We simulate the ribosome atom our simulations reveal the atomistic National Science Foundation of powerful supercomputers together with for atom as well as the surrounding details of how this goes down. innovative imaging modalities to address environment, including several million COLLABORATORS the molecular mechanisms behind the atoms. The ribosome is constantly in How would you explain, in layman’s • Scott Blanchard, Weill Cornell Medical accommodation or rejection of tRNA by motion due to collisions with water terms, the concept of ‘bio-inspired College mRNA, right down to the atomic level. molecules and other factors. When the nanoscale computers’? • Wataru Nishima, New Mexico Once the correct tRNA is accepted by time is right, it undergoes large changes The ribosome is one of the only Consortium the ribosome, the tRNA must navigate in its structure, similar to a fashion model molecular machines in the cell that • Kara Fischer, New Mexico Consortium through the complicated crevices inside striking different poses, or a martial actually processes information (it’s • Christian Spahn, Charite the ribosome so it can escape and make artist adopting different stances. For the certainly the only one that processes Universitätsmedizin Berlin room for the next incoming tRNA. This ribosome, the succession of different info in a complex way). Specifically, it • Jonathan Dinman, University of process, called translocation, is one of the stances helps shuttle the cargo (transfer performs a ‘look-up table’ operation, an Maryland most complicated in protein synthesis and RNA) through the ribosome. By solving operation widespread in computer remains an enigma at the molecular level. Newton’s equations for each atom programming. Given a three-letter BIO The realisation of this objective will offer several million times, we are able to RNA codon on the messenger Dr Sanbonmatsu has been a a greater insight regarding the origin of simulate this process. We work closely RNA, the ribosome selects the principal investigator at Los the genetic code and, ultimately, of life with Scott Blanchard (Cornell) to compare amino acid corresponding to this Alamos National Laboratory itself. More specifically, these molecular with his single molecule fluorescence codon. In this manner, the ribosome since 2001. She received her BA simulations focus on the ribosome head High resolution cryogenic electron microscopy (cryo-EM) is an emerging field, revolutionising structural biology. experiments of the ribosome and with converts the four-letter language in from Columbia College swivel, the rate-limiting step of tRNA Grey surfaces are based on cryo-EM images of the ribosome from Christian Spahn, a leader in cryo-EM ribosome Christian Spahn (Charite) to ensure of nucleic acids (DNA and RNA) to the at and PhD in translocation through the ribosome. In science. Colored ribbons beneath grey surfaces, are atomic models from the Sanbonmatsu group. Atomic consistency with his cryo-EM structures. 20-letter language of proteins using a Astrophysical, Planetary and Atmospheric models are produced from computer simulations of the ribosome, integrating the cryo-EM information. The this key movement, the ‘head’ of the result is an atomistic view of the cryo-EM study, enabling detailed insights into the mechanism of the ribosome We are working with Jonathan Dinman look-up table of the ensemble of tRNAs Sciences from the University of Colorado small subunit pivots around its neck, over (e.g., head swivel). Upper panels show the ribosome before head swivel; lower panels, after head swivel. Lower (Maryland) to calibrate our computer (~40 tRNAs in bacteria). The size of the at Boulder. In 2012, she was elected the body of the small subunit, moving right, final atomistic model is consistent with cryo-EM grey surface. Magenta, large subunit RNA; pink, large simulations with biochemical studies of ribosome is ~20 nm. In this sense, the fellow to the American Physical Society subunit proteins; blue, small subunit RNA; cyan, small subunit proteins. the mRNA and tRNA by exactly one local motions of the ribosome. ribosome is a nanoscale computer. One for 'pioneering the computer simulation codon. The next codon is then exposed, could imagine rigging the ribosome to of molecular machines and biomolecular ready to bind with the next tRNA. The the flexibility with which the rRNA can be essential for the translocation of tRNA through Provided that your research perform other calculations. complexes’. purpose of these simulations is to create chemically modified – a process that has ribosomes. Translocation is catalysed by an successfully explains the molecular efficient predictions of the respective long been used in order to probe ribosome elongation factor that dictates the precise mechanism behind tRNA translocation, Which factors do you think are the CONTACT energy landscape of the head swivel, structure and function. The prospect of movement of both the two tRNAs and the what will the implications be in both most important in order to develop Dr Karissa Sanbonmatsu thus identifying regions of the ribosome creating well-defined datasets of rRNA mRNA within the ribosome. If we can better research and industry? comprehensive datasets of rRNA base New Mexico Consortium that place constraints on the maximal base flexibilities is key to understanding the understand how this process works then we Scott Blanchard (Cornell) is identifying flexibilities? 4200 West Jemez Rd., Suite 301 displacements of the head. dynamics of the ribosome. Excitingly, the open the door to understanding both how our a potential emerging class of antibiotics Recently, Illumina sequencing has Los Alamos, NM 87544 current work performed by the Sanbonmatsu own ribosomes work and how we may be able that target ribosome dynamics. While enabled high throughput SHAPE USA FLEXIBILITY, ELONGATION AND group in collaboration with Jonathan Dinman to target ribosomal activity in pathogens. over 50% of antibiotics used in the US probing on the genome-wide scale, FLUCTUATION (University of Maryland) has already revealed target the ribosome, most block tRNA especially in the field of long non-coding E: kys@lanl. gov Ribosomes are made up of ribosomal the existence of fluctuation hotspots. Such Conclusively, Dr Sanbonmatsu’s pioneering binding or clog up the ribosome tunnel, RNA in and development. W: http://sanbonmatsu.org/ RNA (rRNA) and proteins. A great part of fluctuation hotspots have the capacity to allow research has already provided the scientific preventing the nascent protein from Performing SHAPE-seq on ribosomes http://www.lanl.gov/science-innovation/ Dr Sanbonmatsu’s research focuses on large scale conformation changes that are community with a greater insight into the escaping. Blanchard is using single in many species would help provide features/faces-of-science/karissa- underlying mechanisms of a diverse array molecule studies to identify antibiotics interesting data. Jonathan Dinman has sanbonmatsu.php of non-coding RNA systems. Her special that stop the ribosome from shifting made great strides in this direction. interest in ribosomes and her promising from stance to stance. If you think of the The prospect of creating well-defined findings set the stage for joint computational ribosome as an automobile engine, the What are your hopes for the future of and experimental studies that could lead to antibiotic would be a monkey wrench, your research? datasets of rRNA base flexibilities is breakthroughs in the development of bio- grinding the ribosome to a halt. While the We hope to uncover the quantitative inspired nanoscale computers but also allow single molecule experiments show the energy landscape of translocation. key to understanding the dynamics of for a greater insight regarding the origin of the ribosome life itself.

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