Optimizing eRF1 to Enable the Genetic Encoding of Three Distinct Noncanonical Amino Acids in Mammalian Cells.

Site-specific incorporation of distinct noncanonical amino acids (ncAAs) into proteins via genetic code expansion in mammalian cells represents a new avenue for protein engineering. Reassigning three TAGs with the same ncAA in mammalian cells has previously been achieved using translational machinery. However, simultaneous recoding of three nonsense codons with distinct ncAAs in mammalian cells remains a challenge due to low incorporation efficiencies. Here, three optimized aaRS/tRNA pairs (i.e., the E. coli-derived tyrosyl (EcTyr)/tRNAUUA , E. coli-derived leucyl (EcLeu)/tRNACUA , and Methanosarcina mazei pyrrolysyl (MmPyl)/tRNAUCA pairs) are screened for ncAA incorporation. Furthermore, introduced combinations of eukaryotic release factor 1 (eRF1) mutants (E55R, E55D, N129D, and Y125F) significantly improve the encoding efficiency of the three premature stop codons' sites from 0.78% to 11.6%. Thus, site-specific incorporation of three distinct ncAAs into a single protein is achieved in this study. This work markedly expands the potential for multiple site-specific protein modifications within mammalian cells, thereby facilitating new in vivo applications.

Expression of the translation termination factor eRF1 is autoregulated by translational readthrough and 3'UTR intron-mediated NMD in Neurospora crassa.

Eukaryotic release factor 1 (eRF1) is a translation termination factor that binds to the ribosome at stop codons. The expression of eRF1 is strictly controlled, since its concentration defines termination efficiency and frequency of translational readthrough. Here, we show that eRF1 expression in Neurospora crassa is controlled by an autoregulatory circuit that depends on the specific 3'UTR structure of erf1 mRNA. The stop codon context of erf1 promotes readthrough that protects the mRNA from its 3'UTR-induced nonsense-mediated mRNA decay (NMD). High eRF1 concentration leads to inefficient readthrough, thereby allowing NMD-mediated erf1 degradation. We propose that eRF1 expression is controlled by similar autoregulatory circuits in many fungi and seed plants and discuss the evolution of autoregulatory systems of different translation termination factors.

Overcoming Near-Cognate Suppression in a Release Factor 1-Deficient Host with an Improved Nitro-Tyrosine tRNA Synthetase.

Genetic code expansion (GCE) technologies incorporate non-canonical amino acids (ncAAs) into proteins at amber stop codons. To avoid unwanted truncated protein and improve ncAA-protein yields, genomically recoded strains of Escherichia coli lacking Release Factor 1 (RF1) are becoming increasingly popular expression hosts for GCE applications. In the absence of RF1, however, endogenous near-cognate amber suppressing tRNAs can lead to contaminating protein forms with natural amino acids in place of the ncAA. Here, we show that a second-generation amino-acyl tRNA synthetase (aaRS)/tRNACUA pair for site-specific incorporation of 3-nitro-tyrosine could not outcompete near-cognate suppression in an RF1-deficient expression host and therefore could not produce homogenously nitrated protein. To resolve this, we used Rosetta to target positions in the nitroTyr aaRS active site for improved substrate binding, and then constructed of a small library of variants to subject to standard selection protocols. The top selected variant had an ~2-fold greater efficiency, and remarkably, this relatively small improvement enabled homogeneous incorporation of nitroTyr in an RF1-deficient expression host and thus eliminates truncation issues associated with typical RF1-containing expression hosts. Structural and biochemical data suggest the aaRS efficiency improvement is based on higher affinity substrate binding. Taken together, the modest improvement in aaRS efficiency provides a large practical impact and expands our ability to study the role protein nitration plays in disease development through producing homogenous, truncation-free nitroTyr-containing protein. This work establishes Rosetta-guided design and incremental aaRS improvement as a viable and accessible path to improve GCE systems challenged by truncation and/or near-cognate suppression issues.

Cell-Free Protein Synthesis Using S30 Extracts from Escherichia coli RFzero Strains for Efficient Incorporation of Non-Natural Amino Acids into Proteins.

Cell-free protein synthesis is useful for synthesizing difficult targets. The site-specific incorporation of non-natural amino acids into proteins is a powerful protein engineering method. In this study, we optimized the protocol for cell extract preparation from the Escherichia coli strain RFzero-iy, which is engineered to lack release factor 1 (RF-1). The BL21(DE3)-based RFzero-iy strain exhibited quite high cell-free protein productivity, and thus we established the protocols for its cell culture and extract preparation. In the presence of 3-iodo-l-tyrosine (IY), cell-free protein synthesis using the RFzero-iy-based S30 extract translated the UAG codon to IY at various sites with a high translation efficiency of >90%. In the absence of IY, the RFzero-iy-based cell-free system did not translate UAG to any amino acid, leaving UAG unassigned. Actually, UAG was readily reassigned to various non-natural amino acids, by supplementing them with their specific aminoacyl-tRNA synthetase variants (and their specific tRNAs) into the system. The high incorporation rate of our RFzero-iy-based cell-free system enables the incorporation of a variety of non-natural amino acids into multiple sites of proteins. The present strategy to create the RFzero strain is rapid, and thus promising for RF-1 deletions of various E. coli strains genomically engineered for specific requirements.

Dissecting the Contribution of Release Factor Interactions to Amber Stop Codon Reassignment Efficiencies of the Methanocaldococcus jannaschii Orthogonal Pair.

Non-canonical amino acids (ncAAs) are finding increasing use in basic biochemical studies and biomedical applications. The efficiency of ncAA incorporation is highly variable, as a result of competing system composition and codon context effects. The relative quantitative contribution of the multiple factors affecting incorporation efficiency are largely unknown. This manuscript describes the use of green fluorescent protein (GFP) reporters to quantify the efficiency of amber codon reassignment using the Methanocaldococcus jannaschii orthogonal pair system, commonly employed for ncAA incorporation, and quantify the contribution of release factor 1 (RF1) to the overall efficiency of amino acid incorporation. The efficiencies of amber codon reassignments were quantified at eight positions in GFP and evaluated in multiple combinations. The quantitative contribution of RF1 competition to reassignment efficiency was evaluated through comparisons of amber codon suppression efficiencies in normal and genomically recoded Escherichia coli strains. Measured amber stop codon reassignment efficiencies for eight single stop codon GFP variants ranged from 51 to 117% in E. coli DH10B and 76 to 104% in the RF1 deleted E. coli C321.ΔA.exp. Evaluation of efficiency changes in specific sequence contexts in the presence and absence of RF1 suggested that RF1 specifically interacts with +4 Cs and that the RF1 interactions contributed approximately half of the observed sequence context-dependent variation in measured reassignment efficiency. Evaluation of multisite suppression efficiencies suggests that increasing demand for translation system components limits multisite incorporation in cells with competing RF1.

Cell-Free Protein Synthesis for Multiple Site-Specific Incorporation of Noncanonical Amino Acids Using Cell Extracts from RF-1 Deletion E. coli Strains.

Cell-free protein synthesis (CFPS) is an effective method for the site-specific incorporations of noncanonical amino acids (ncAAs) into proteins. The nature of in vitro synthesis enables the use of experimental conditions that are toxic or reduce cellular uptake during in vivo site-specific incorporations of ncAAs. Using the Escherichia coli cell extract (S30) from the highly reproductive RF-1 deletion strains, B-60.∆A::Z and B-95.∆A, with orthogonal tRNA and aminoacyl-tRNA synthetase (aaRS) pairs from Methanosarcina mazei, we have developed CFPS methods for the highly productive and efficient multiple incorporation of ncAAs. In this chapter, we describe our methods for the preparation of the S30 and the orthogonal tRNAPyl and PylRS pair, and two CFPS protocols for ncAA incorporation.

Improving cell-free protein synthesis through genome engineering of Escherichia coli lacking release factor 1.

Site-specific incorporation of non-standard amino acids (NSAAs) into proteins opens the way to novel biological insights and applications in biotechnology. Here, we describe the development of a high yielding cell-free protein synthesis (CFPS) platform for NSAA incorporation from crude extracts of genomically recoded Escherichia coli lacking release factor 1. We used genome engineering to construct synthetic organisms that, upon cell lysis, lead to improved extract performance. We targeted five potential negative effectors to be disabled: the nuclease genes rna, rnb, csdA, mazF, and endA. Using our most productive extract from strain MCJ.559 (csdA(-) endA(-)), we synthesized 550±40 µg mL(-1) of modified superfolder green fluorescent protein containing p-acetyl-L-phenylalanine. This yield was increased to ∼1300 µg mL(-1) when using a semicontinuous method. Our work has implications for using whole genome editing for CFPS strain development, expanding the chemistry of biological systems, and cell-free synthetic biology.

A bacterial strain with a unique quadruplet codon specifying non-native amino acids.

The addition of noncanonical amino acids to the genetic code requires unique codons not assigned to the 20 canonical amino acids. Among the 64 triplet codons, only the three nonsense "stop" codons have been used to encode non-native amino acids. Use of quadruplet "frame-shift" suppressor codons provides an abundant alternative but suffers from low suppression efficiency as a result of competing recognition of their first three bases by endogenous host tRNAs or release factors. Deletion of release factor 1 in a genomically recoded strain of E. coli (E. coli C321), in which all endogenous amber stop codons (UAG) are replaced with UAA, abolished UAG mediated translation termination. Here we show that a Methanocaldococcus jannaschii-derived frame-shift suppressor tRNA/aminoacyl-tRNA synthetase pair enhanced UAGN suppression efficiency in this recoded bacterial strain. These results demonstrate that efficient quadruplet codons for encoding non-native amino acids can be generated by eliminating competing triplet codon recognition at the ribosome.

Unnatural amino acid incorporation in E. coli: current and future applications in the design of therapeutic proteins.

Unnatural amino acid (UAA) incorporation by amber codon suppression offers scientists a powerful tool to modify the properties of proteins at will. UAA incorporation has been used for a plethora of fundamental research applications and, more recently, also for the selective modification of therapeutic proteins. In this review most recent developments in Escherichia coli codon expansion and, unnatural amino acid incorporation are discussed together with some remarkable recent developments in improved efficient UAA incorporation. We focus on the generation of proteins that hold promise for future therapeutic applications that would be impossible to obtain without unnatural amino acid incorporation, including the generation of bi-specific antibodies and antibody drug conjugates.

Protein-RNA recognition: cracking the code.

In early papers, the intent was to find a simple protein-RNA/DNA recognition code. Many people expected a one-to-one correspondence between amino acids and nucleic bases, similar to the code that specifies how one DNA base pairs with another. Despite the lack of such a code, which was evident in the first crystal structures, researchers were indeed unwilling to give up on the idea. Despite the intense interest, a simple one-to-one correspondence has not materialised. The work presented here revisits this theme, and reports a general trend in which four elementary amino acids - G, A, V, and D - have a specific selectivity for four basic nucleotides - g, c, u, and a. During the evolution, as amino acid alphabets increased, new amino acids substituted G, A, V, D amino acids in way to keep hydropathic similarity and the selectivity to minimise errors in established RNA-protein interactions, 1-letter code was created. Additionally, the first nucleotide in codons is used for a 2-letter code. Protein-RNA recognition, visualised by these two code principles, uses a rotation of sensing and anti-sensing sequences in architecture of recognising peptides.

Overexpression, crystallization and preliminary X-ray crystallographic analysis of release factor eRF1-1 from Arabidopsis thaliana.

Peptide release factor 1 (RF1) regulates the termination of translation in protein synthesis by recognizing the stop codons. The eukaryotic RF1s (eRF1s) from Arabidopsis thaliana and human have different stop-codon preferences even though they share high sequence similarity. Based on known RF1 structures, it has been suggested that the specificity depends on both the local structure and the domain arrangement, but the lack of a structure of Arabidopsis eRF1 hinders a detailed comparison. To reveal the mechanism of stop-codon recognition and compare it with that of human eRF1, one of the three Arabidopsis eRF1s, AteRF1-1, was studied and a preliminary X-ray crystallographic analysis is reported here. The protein was overexpressed in Escherichia coli and crystallized at room temperature using the vapour-diffusion method. Crystals were grown from 1.6 M lithium sulfate, 0.1 M Tris-HCl pH 8.0, 2%(v/v) PEG 400 and diffracted to 3.77 Šresolution. The data were processed in point group 622, with unit-cell parameters a = b = 136.6, c = 325.7 Å.