Multiplex suppression of four quadruplet codons via tRNA directed evolution.

Genetic code expansion technologies supplement the natural codon repertoire with assignable variants in vivo, but are often limited by heterologous translational components and low suppression efficiencies. Here, we explore engineered Escherichia coli tRNAs supporting quadruplet codon translation by first developing a library-cross-library selection to nominate quadruplet codon-anticodon pairs. We extend our findings using a phage-assisted continuous evolution strategy for quadruplet-decoding tRNA evolution (qtRNA-PACE) that improved quadruplet codon translation efficiencies up to 80-fold. Evolved qtRNAs appear to maintain codon-anticodon base pairing, are typically aminoacylated by their cognate tRNA synthetases, and enable processive translation of adjacent quadruplet codons. Using these components, we showcase the multiplexed decoding of up to four unique quadruplet codons by their corresponding qtRNAs in a single reporter. Cumulatively, our findings highlight how E. coli tRNAs can be engineered, evolved, and combined to decode quadruplet codons, portending future developments towards an exclusively quadruplet codon translation system.

Cross-species conservation of complementary amino acid-ribonucleobase interactions and their potential for ribosome-free encoding.

The role of amino acid-RNA nucleobase interactions in the evolution of RNA translation and protein-mRNA autoregulation remains an open area of research. We describe the inference of pairwise amino acid-RNA nucleobase interaction preferences using structural data from known RNA-protein complexes. We observed significant matching between an amino acid's nucleobase affinity and corresponding codon content in both the standard genetic code and mitochondrial variants. Furthermore, we showed that knowledge of nucleobase preferences allows statistically significant prediction of protein primary sequence from mRNA using purely physiochemical information. Interestingly, ribosomal primary sequences were more accurately predicted than non-ribosomal sequences, suggesting a potential role for direct amino acid-nucleobase interactions in the genesis of amino acid-based ribosomal components. Finally, we observed matching between amino acid-nucleobase affinities and corresponding mRNA sequences in 35 evolutionarily diverse proteomes. We believe these results have important implications for the study of the evolutionary origins of the genetic code and protein-mRNA cross-regulation.

Functional Class I and II Amino Acid-activating Enzymes Can Be Coded by Opposite Strands of the Same Gene.

Aminoacyl-tRNA synthetases (aaRS) catalyze both chemical steps that translate the universal genetic code. Rodin and Ohno offered an explanation for the existence of two aaRS classes, observing that codons for the most highly conserved Class I active-site residues are anticodons for corresponding Class II active-site residues. They proposed that the two classes arose simultaneously, by translation of opposite strands from the same gene. We have characterized wild-type 46-residue peptides containing ATP-binding sites of Class I and II synthetases and those coded by a gene designed by Rosetta to encode the corresponding peptides on opposite strands. Catalysis by WT and designed peptides is saturable, and the designed peptides are sensitive to active-site residue mutation. All have comparable apparent second-order rate constants 2.9-7.0E-3 M(-1) s(-1) or ∼750,000-1,300,000 times the uncatalyzed rate. The activities of the two complementary peptides demonstrate that the unique information in a gene can have two functional interpretations, one from each complementary strand. The peptides contain phylogenetic signatures of longer, more sophisticated catalysts we call Urzymes and are short enough to bridge the gap between them and simpler uncoded peptides. Thus, they directly substantiate the sense/antisense coding ancestry of Class I and II aaRS. Furthermore, designed 46-mers achieve similar catalytic proficiency to wild-type 46-mers by significant increases in both kcat and Km values, supporting suggestions that the earliest peptide catalysts activated ATP for biosynthetic purposes.

Translation attenuation via 3' terminal codon usage in bovine csn1s2 is responsible for the difference in αs2- and ß-casein profile in milk.

The rate of secretion of αs2-casein into bovine milk is approximately 25% of that of ß-casein, yet mammary expression of their respective mRNA transcripts (csn1s2 and csn2) is not different. Our objective was to identify molecular mechanisms that explain the difference in translation efficiency between csn1s2 and csn2. Cell-free translational efficiency of csn2 was 5 times that of csn1s2. Transcripts of csn1s2 distributed into heavier polysomes than csn2 transcripts, indicating an attenuation of elongation and/or termination. Stimulatory and inhibitory effects of the 5' and 3' UTRs on translational efficiency were different with luciferase and casein sequences in the coding regions. Substituting the 5' and 3' UTRs from csn2 into csn1s2 did not improve csn1s2 translation, implicating the coding region itself in the translation difference. Deletion of a 28-codon fragment from the 3' terminus of the csn1s2 coding region, which displays codons with low correlations to cell fitness, increased translation to a par with csn2. We conclude that the usage of the last 28 codons of csn1s2 is the main regulatory element that attenuates its expression and is responsible for the differential translational expression of csn1s2 and csn2.

Lys(203) and Lys(382) are essential for the proteasomal degradation of BACE1.

Amyloid ß protein (Aß) is the primary component of senile plaques in Alzheimer's disease brains and its aggregate form is neurotoxic. Aß is generated through proteolysis of ß-amyloid precursor protein (APP) by two proteases: ß-secretase and γ-secretase. BACE1, the ß-secretase in vivo and the key rate-limiting enzyme that initiates the formation of Aß, is an attractive drug target for AD therapy. Our previous study demonstrated that BACE1 is ubiquitinated and its degradation and effect on APP cleaving process are mediated by the ubiquitin-proteasome pathway. However, the specific underlying mechanism is still not well defined. In present study, we determined the specific binding sites responsible for the proteasomal degradation of BACE1. Ten fragments of human BACE1 cDNA with each of them containing 1 to 3 Lys codons were cloned, and HEK293 cells transfected with these recombinant plasmids were treated with specific proteasome inhibitor lactacystin. The protein levels of fragment-3 (Pro(149)-Leu(180)), -4 (IIe(179)-Ser(230)) and -8 (Met(349)-Arg(400)) were significantly increased by lactacystin treatment, and immunocytochemical staining results showed that fragment-3, -4 and -8 proteins were colocalized with ubiquitin. Site-directed mutagenesis at Lys(203) and Lys(382) of BACE1 abolished the proteasomal degradation of BACE1 and affected APP processing at ß site and Aß production. Taken together, our study demonstrated that BACE1 Lys(203) and Lys(382) are essential for its proteasomal degradation, and the results may advance our understanding of regulation of BACE1 and APP processing by the ubiquitin proteasome system in AD pathogenesis and shed new insights on its pharmaceutical potential.

S-Adenosylmethionine induces BldH and activates secondary metabolism by involving the TTA-codon control of bldH expression in Streptomyces lividans.

In the present study, a mechanism for S-adenosylmethionine (SAM) to promote secondary metabolism was characterized in terms of bldH sl) expression in Streptomyces lividans. A previous study demonstrated that SAM, on application at 2 microM, induces the transcription of the strR promoter (strRp), which originates from Streptomyces griseus, in S. lividans. An inactivation study verified that bldH sl is essential to strRp transcription in S. lividans and it was demonstrated that the effects of SAM on the induction of strRp activity, on the transcription of redZ and actII-orf4, and on antibiotic production were compromised when the unique leucine TTA-codon of bldH sl was changed to TTG. Western blot analysis revealed that SAM supplementation enhances the expression of bldH sl when the TTA-codon was intact but not when the TTG replacement was provided. This study validates the involvement of BldH sl in the potentiating effect of SAM on the antibiotic production and substantiates that SAM controls the expression of bldH sl through the TTA-codon control in translating bldH mRNA. It is argued here that the intracellular SAM-level modulates the maturation of bldA, which encodes the UUA-codon tRNA and controls secondary metabolism in S. lividans.

The Proteomic Code: a molecular recognition code for proteins.

BACKGROUND: The Proteomic Code is a set of rules by which information in genetic material is transferred into the physico-chemical properties of amino acids. It determines how individual amino acids interact with each other during folding and in specific protein-protein interactions. The Proteomic Code is part of the redundant Genetic Code. REVIEW: The 25-year-old history of this concept is reviewed from the first independent suggestions by Biro and Mekler, through the works of Blalock, Root-Bernstein, Siemion, Miller and others, followed by the discovery of a Common Periodic Table of Codons and Nucleic Acids in 2003 and culminating in the recent conceptualization of partial complementary coding of interacting amino acids as well as the theory of the nucleic acid-assisted protein folding. METHODS AND CONCLUSIONS: A novel cloning method for the design and production of specific, high-affinity-reacting proteins (SHARP) is presented. This method is based on the concept of proteomic codes and is suitable for large-scale, industrial production of specifically interacting peptides.

P-site pairing subtleties revealed by the effects of different tRNAs on programmed translational bypassing where anticodon re-pairing to mRNA is separated from dissociation.

Programmed ribosomal bypassing occurs in decoding phage T4 gene 60 mRNA. Half the ribosomes bypass a 50 nucleotide gap between codons 46 and 47. Peptidyl-tRNA dissociates from the "take-off" GGA, codon 46, and re-pairs to mRNA at a matched GGA "landing site" codon directly 5' of codon 47 where translation resumes. The system described here allows the contribution of peptidyl-tRNA re-pairing to be measured independently of dissociation. The matched GGA codons have been replaced by 62 other matched codons, giving a wide range of bypassing efficiencies. Codons with G or C in either or both of the first two codon positions yielded high levels of bypassing. The results are compared with those from a complementary study of non-programmed bypassing, where the combined effects of peptidyl-tRNA dissociation and reassociation were measured. The wild-type, GGA, matched codons are the most efficient in their gene 60 context in contrast to the relatively low value in the non-programmed bypassing study.

[Selenoproteins--atypical function of the UGA codon]. / Selenoproteiny--nietypowa funkcja kodonu UGA.

Selenium is an essential trace element for animals and humans. It's a structural component of the active center of several important mammalian enzymes, like gluthatione peroxidase (GSH-Px), iodothyronine deiodinase, and others selenoproteins. Paper describes selenium functions in physiological states, and mechanism of incorporation of selenocysteine into selenoproteins. This incorporation requires the alternative decoding of UGA, which typically serves as a stop codon. The most important element is the secondary structure of mRNA named SECIS (selenocysteine inserstion sequence).

Synthetic cDNA encoding the rat AT1a receptor: a useful tool for structure-function relationship analysis.

To carry out systematic structure-function studies of the rat angiotensin II receptors by site directed mutagenesis, or production of chimeric receptors, we have produced a synthetic cDNA coding for the AT1a receptor. The synthetic cDNA is 1101 base pairs long, and contains 49 unique restriction sites that are on the average 23 base pairs apart, allowing replacement of specific restriction fragments by synthetic counterparts containing the desired modified sequence. The total cDNA was assembled in the expression vector pECE. After stable expression in Chinese Hamster Ovary cells, the protein encoded by this synthetic cDNA presents a pharmacological profile and a signal transduction mechanism indistinguishable from the wild type rat AT1a receptor.

Amino acid complementarity: testing of hypotheses.

To determine whether hypotheses about the complementarity of amino acids based on the genetic code reflect the amino acid contact preferences found in natural proteins, the average contact probabilities for hypothetical complementary amino acid pairs were compared with those for all possible remaining pairs of the corresponding subset. A statistically significant preference was found for contact between amino acids with codons which had the same central nucleotide. Conversely, the contact probabilities for amino acids with complementary codons either did not exceed, or exceeded only insignificantly, the value for the corresponding remainder subset. The data obtained do not support the hypothesis for the complementarity of peptides coded by complementary RNA strands.