Unlike equilibrium statistical mechanics, with its well-established foundations, a similar widely accepted framework for non-equilibrium statistical mechanics (NESM) remains elusive. Here, we review some of the many recent activities on NESM, focusing on some of the fundamental issues and general aspects. Using the language of stochastic Markov processes, we emphasize general properties of the evolution of configurational probabilities, as described by master equations. Of particular interest are systems in which the dynamics violates detailed balance, since such systems serve to model a wide variety of phenomena in nature. We next review two distinct approaches for investigating such problems. One approach focuses on models sufficiently simple to allow us to find exact, analytic, non-trivial results. We provide detailed mathematical analyses of a one-dimensional continuous-time lattice gas, the totally asymmetric exclusion process. It is regarded as a paradigmatic model for NESM, much like the role the Ising model played for equilibrium statistical mechanics. It is also the starting point for the second approach, which attempts to include more realistic ingredients in order to be more applicable to systems in nature. Restricting ourselves to the area of biophysics and cellular biology, we review a number of models that are relevant for transport phenomena. Successes and limitations of these simple models are also highlighted.

/pdf/non-equilibrium-statistical-mechanics-from-a-paradigmatic-1gq17no141.pdf

Non-equilibrium statistical mechanics: from a paradigmatic model to biological transport

Unlike equilibrium statistical mechanics, with its well-established foundations, a similar widely-accepted framework for non-equilibrium statistical mechanics (NESM) remains elusive. Here, we review some of the many recent activities on NESM, focusing on some of the fundamental issues and general aspects. Using the language of stochastic Markov processes, we emphasize general properties of the evolution of configurational probabilities, as described by master equations. Of particular interest are systems in which the dynamics violate detailed balance, since such systems serve to model a wide variety of phenomena in nature. We next review two distinct approaches for investigating such problems. One approach focuses on models sufficiently simple to allow us to find exact, analytic, non-trivial results. We provide detailed mathematical analyses of a one-dimensional continuous-time lattice gas, the totally asymmetric exclusion process (TASEP). It is regarded as a paradigmatic model for NESM, much like the role the Ising model played for equilibrium statistical mechanics. It is also the starting point for the second approach, which attempts to include more realistic ingredients in order to be more applicable to systems in nature. Restricting ourselves to the area of biophysics and cellular biology, we review a number of models that are relevant for transport phenomena. Successes and limitations of these simple models are also highlighted.

Non-equilibrium statistical mechanics: From a paradigmatic model to biological transport

Stochastic mechano-chemical kinetics of molecular motors: A multidisciplinary enterprise from a physicist’s perspective

Owing to the degeneracy of the genetic code, a protein sequence can be encoded by many different synonymous mRNA coding sequences. Synonymous codon usage was once thought to be functionally neutral, but evidence now indicates it is shaped by evolutionary selection and affects other aspects of protein biogenesis beyond specifying the amino acid sequence of the protein. Synonymous rare codons, once thought to have only negative impacts on the speed and accuracy of translation, are now known to play an important role in diverse functions, including regulation of cotranslational folding, covalent modifications, secretion, and expression level. Mutations altering synonymous codon usage are linked to human diseases. However, much remains unknown about the molecular mechanisms connecting synonymous codon usage to efficient protein biogenesis and proper cell physiology. Here we review recent literature on the functional effects of codon usage, including bioinformatics approaches aimed at identifying general roles for synonymous codon usage.

Roles for Synonymous Codon Usage in Protein Biogenesis

The sequential addition of amino acids to a growing polypeptide chain is carried out by the ribosome in a complicated multistep process called the elongation cycle. It involves accurate selection of each aminoacyl tRNA as dictated by the mRNA codon, catalysis of peptide bond formation, and movement of the tRNAs and mRNA through the ribosome. The process requires the GTPase factors elongation factor Tu (EF-Tu) and EF-G. Not surprisingly, large conformational changes in both the ribosome and its tRNA substrates occur throughout protein elongation. Major advances in our understanding of the elongation cycle have been made in the past few years as a result of high-resolution crystal structures that capture various states of the process, as well as biochemical and computational studies.

Structural basis of the translational elongation cycle.

https://www.cell.com/molecular-cell/pdf/S1097-2765(11)00257-7.pdf

Single-molecule fluorescence measurements of ribosomal translocation dynamics.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/j.febslet.2013.10.016

Engine out of the chassis: Cell-free protein synthesis and its uses

Pauses regulate the rhythm of ribosomal protein synthesis. Mutations disrupting even minor pauses can give rise to improperly formed proteins and human disease. Such minor pauses are difficult to characterize by ensemble methods, but can be readily examined by single-molecule (sm) approaches. Here we use smFRET to carry out real-time monitoring of the expression of a full-length protein, the green fluorescent protein variant Emerald GFP. We demonstrate significant correlations between measured elongation rates and codon and isoacceptor tRNA usage, and provide a quantitative estimate of the effect on elongation rate of replacing a codon recognizing an abundant tRNA with a synonymous codon cognate to a rarer tRNA. Our results suggest that tRNA selection plays an important general role in modulating the rates and rhythms of protein synthesis, potentially influencing simultaneous co-translational processes such as folding and chemical modification.

Quantifying elongation rhythm during full-length protein synthesis

We present a flexible, real-time-coupled transcription-translation assay that involves the continuous monitoring of fluorescent Emerald GFP formation. Along with numerical simulation of a reaction kinetics model, the assay permits quantitative estimation of the effects on full-length protein synthesis of various additions, subtractions or substitutions to the protein synthesis machinery. Since the assay uses continuous fluorescence monitoring, it is much simpler and more rapid than other assays of protein synthesis and is compatible with high-throughput formats. Straightforward alterations of the assay permit determination of (i) the fraction of ribosomes in a cell-free protein synthesis kit that is active in full-length protein synthesis and (ii) the relative activities in supporting protein synthesis of modified (e.g. mutated, fluorescent-labeled) exogenous components (ribosomes, amino acid-specific tRNAs) that replace the corresponding endogenous components. Ribosomes containing fluorescent-labeled L11 and tRNAs labeled with fluorophores in the D-loop retain substantial activity. In the latter case, the extent of activity loss correlates with a combination of steric bulk and hydrophobicity of the fluorophore.

/pdf/real-time-assay-for-testing-components-of-protein-synthesis-4rdnnucwl3.pdf

Real-time assay for testing components of protein synthesis

Continued dramatic progress in the elucidation of the structures of the bacterial ribosome and its functional complexes has led to proposals for the detailed mechanisms of ribosome-catalyzed protein synthesis (Schmeing and Ramakrishnan, 2009; Agirrezabala and Frank, 2009). Ensemble rapid reaction kinetics (Antoun et al., 2006; Daviter et al., 2006; Dorner et al., 2006; Grigoriadou et al., 2007; Hetricket al., 2009; Pan et al., 2007, 2008; Pape et al., 1998; Phelps and Joseph 2006; Rodnina et al., 1997; Savelsbergh et al., 2003; Walker et al., 2008; Wintermeyer et al., 2004; Zaher and Green, 2009; Zavialov and Ehrenberg, 2003) and single-molecule (Blanchard et al., 2004a, b; Cornish et al., 2008, 2009; Fei et al., 2008, 2009; Marshall et al., 2008, 2009; Munro et al., 2007, 2010a, b; Uemura et al., 2010; Wang et al., 2007) studies of the translational machinery in the past several years have resulted in increased understanding of many aspects of the initiation, elongation, and termination phases of protein synthesis, but many essential points remain to be elucidated.

Gabriel Rosenblum

Papers

Single-molecule fluorescence measurements of ribosomal translocation dynamics.

Engine out of the chassis: Cell-free protein synthesis and its uses

Quantifying elongation rhythm during full-length protein synthesis

Real-time assay for testing components of protein synthesis

Mechanism and dynamics of the elongation cycle