Nucleic Acids Research最新文献

The single subunit RNA polymerases (ssRNAPs) of bacteriophages are highly interesting targets for the prediction and engineering of specific protein-DNA interactions. Despite extensive existing studies focusing on particular ssRNAPs such as the T7 RNAP, few rules governing the protein-DNA sequence covariations across diverse ssRNAPs and their cognate promoters are clearly known. Here, aiming to reveal such rules, we comprehensively mined promoters of various categories of ssRNAPs from phage genomes. For T7-like RNAPs, direct coupling analyses of the predicted set of RNAP-promoter pairs revealed that the interaction specificity was dominantly encoded by the amino acid and nucleotide residues at only a few key positions. The covariations between the amino acid and the nucleotide residues at these positions were summarized into a sparsely connected network. Using experimentally verified connections in this network, we designed a set of orthogonal T7 RNAP-promoter variants that showed more stringent orthogonality than previously reported sets. We further designed and experimentally verified variants with novel interactions. These results provided guidance for engineering novel RNAP-promoter pairs for synthetic biology or other applications. Our study also demonstrated the use of comprehensive genome mining in combination with sequence covariation analysis in the prediction and engineering of specific protein-DNA interactions.

The average eukaryotic transfer ribonucleic acid (tRNA) contains 13 post-transcriptional modifications; however, their functional impact is largely unknown. Our understanding of the complex tRNA aminoacylation machinery in metazoans also remains limited. Herein, using a series of high-resolution cryo-electron microscopy (cryo-EM) structures, we provide the mechanistic basis for recognition and aminoacylation of fully modified cellular tRNALys3 by human lysyl-tRNA synthetase (h-LysRS). The tRNALys3 anticodon loop modifications S34 (mcm5s2U) and R37 (ms2t6A) play an integral role in recognition by h-LysRS. Modifications in the T-, variable-, and D-loops of tRNALys3 are critical for ordering the metazoan-specific N-terminal domain of LysRS. The two catalytic steps of tRNALys3 aminoacylation are structurally ordered; docking of the 3'-CCA end in the active site cannot proceed until the lysyl-adenylate intermediate is formed and the pyrophosphate byproduct is released. Association of the h-LysRS-tRNALys3 complex with a multi-tRNA synthetase complex-derived peptide shifts the equilibrium toward the 3'-CCA end "docked" conformation and allosterically increases h-LysRS catalytic efficiency. The insights presented here have broad implications for understanding the role of tRNA modifications in protein synthesis, the human aminoacylation machinery, and the growing catalog of metabolic and neurological diseases linked to it.

Protein synthesis is a vital process that is highly regulated at the initiation step of translation. Eukaryotic 5' transcript leaders (TLs) contain a variety of cis-acting features that influence translation and messenger RNA stability. However, the relative influences of these features in natural TLs are poorly characterized. To address this, we used massively parallel reporter assays (MPRAs) to quantify RNA levels, ribosome loading, and protein levels from 11,027 natural yeast TLs in vivo and systematically compared the relative impacts of their sequence features on gene expression. We found that yeast TLs influence gene expression over two orders of magnitude. While a leaky scanning model using Kozak contexts (-4 to +1 around the AUG start) and upstream AUGs (uAUGs) explained half of the variance in expression across TLs, the addition of other features explained ∼80% of gene expression variation. Our analyses detected key cis-acting sequence features, quantified their effects in vivo, and compared their roles to motifs reported from an in vitro study of ribosome recruitment. In addition, our work quantitated the effects of alternative transcription start site usage on gene expression in yeast. Thus, our study provides new quantitative insights into the roles of TL cis-acting sequences in regulating gene expression.

During meiosis, each chromosome pair experiences at least one crossover (CO), which directs their balanced segregation in addition to shuffling genetic information. COs tend to be away from each other, a phenomenon known as CO interference. The main biochemical pathway for CO formation, which is conserved in distant eukaryotes, involves the ZMM proteins together with the MLH1-MLH3 complex (MutLγ). Here, we aim to clarify the role of MutLγ in CO formation in Arabidopsis thaliana. We show that AtMutLγ is partially dispensable for ZMM-dependent CO formation. HEI10 large foci-that mark CO sites in wild-type-form at a normal level in mlh1 and mlh3 mutants, but are inefficiently maturated into COs. Mutating the MUS81 nuclease in either mlh1 or mlh3 leads to chromosome fragmentation, which is suppressed by further mutating the zmm msh5. This suggests that in the absence of MutLγ, recombination intermediates produced by ZMMs are resolved by MUS81, which does not ensure CO formation. Finally, CO interference is marginally affected in mlh1, which is compatible with a random sub-sampling of normally patterned CO sites. We conclude that AtMutLγ imposes designated recombination intermediates to be resolved exclusively as COs, supporting the view that MutLγ asymmetrically resolves double-Holliday junctions, yielding COs.

G-quadruplex (G4) is a guanine-rich secondary structure found in DNA and RNA involved in various biological roles. Recently, a non-canonical RNA G-quadruplex (rG4), known as poly(UG) (pUG) fold, was discovered in Caenorhabditis elegans. This unique structure was found to induce RNA interference (RNAi) upon recruitment of RNA-dependent RNA polymerase (RdRP), resulting in trans-generational gene silencing. Herein, we develop a novel L-RNA aptamer, L-apt3.1, that binds to the pUG fold. We uncover that L-apt3.1 consists of a parallel rG4 structural motif, and mutagenesis analysis illustrates that the rG4 motif in L-apt3.1 is essential for pUG fold recognition. We show that L-apt3.1 interacts strongly with pUG fold, and notably, it is the first reported aptamer that can bind to pUG fold in vitro. We also demonstrate that L-apt3.1 possesses great biostability in cellular environments and negligible toxicity in vivo. Furthermore, we report that L-apt3.1 can interact with pUG fold in vivo, and with a comparable performance to the G4 ligand, N-methyl mesoporphyrin, in inhibiting gene silencing in C. elegans. Overall, we demonstrate the development of pUG fold-targeting L-RNA aptamer for the first time, and show that this new aptamer tool can be applied to control pUG fold-mediated gene expression in vivo.