This study showed that an additional adenosine nucleotide upstream of the terminator hairpin leads to improved protein production while reducing terminator read-through, revealing an important role for the nucleotides directly upstream of the terminator hairpin.
Abstract
Transcriptional termination efficiency is considered an important parameter for finetuning bacterial gene expression. Still, the design principles that determine transcription termination efficiency remain poorly understood. In this study, we aimed to investigate the impact of the 3’ untranslated region (3’UTR) on gene expression in Escherichia coli and other bacteria. First, 3’UTR variant sequences were generated, with randomized 30 bp sequences inserted between the STOP-codon and an intrinsic terminator, consisting of a GC-rich hairpin and a downstream poly(U)-tail. Using three reporter genes, it was found that different 3’UTR sequences resulted in an up to five-fold difference in protein production, independent of the upstream coding sequence. The highest protein production was achieved when an adenosine was present directly upstream of the terminator hairpin. This was consolidated by systematic substitution of key nucleotides of the terminator and assessing their effect on mRNA and protein levels. Subsequently, we developed a predictive random forest machine learning model trained on the termination efficiency of different natural and synthetic terminator sequences, revealing an important role for the nucleotides directly upstream of the terminator hairpin. Altogether, this study showed that an additional adenosine nucleotide upstream of the terminator hairpin leads to improved protein production while reducing terminator read-through. GRAPHICAL ABSTRACT
Slowly translated codons are overrepresented among the first approximately 30 codons of natural genes across all domains of life, yet the functional basis for this conserved feature remains incompletely understood. Using the Escherichia coli lacZ gene as a model, we previously showed that insertion of fast-translated codons into the early coding region dramatically reduces β-galactosidase production by increasing premature transcription termination and decreasing mRNA stability. Here we show that premature transcription termination events occur several nucleotides downstream of the fast-codon inserts, at positions coinciding with previously characterized Rho-dependent intragenic termination sites in lacZ. To exclude the possibility that the specific amino acid sequence of the inserts-rather than their translation speed-was responsible for termination, we constructed two additional lacZ variants with distinct fast-translated sequences, including one replacing the 30 earliest lacZ codons with their fastest synonymous alternatives. Both variants showed premature transcription termination of comparable magnitude to the original inserts, demonstrating that it is the high translation rate itself which causes premature termination. Analysis of a conservative set of seven high-confidence fast-translated codons across natural highly expressed genes and the synthetic constructs revealed that a run of consecutive fast codons is the feature that most clearly distinguishes the terminating sequences from natural genes. We propose that excessively rapid synthesis of the N-terminal part of a polypeptide may impair its proper entry into the ribosomal exit tunnel, thereby disrupting transcription-translation coupling and exposing downstream mRNA to Rho-dependent termination.
S. Pedersen, Alberte Honoré Jepsen, Bertil Gummesson et al.· Molecular Microbiology· 0 citations
This work systematically quantified the intracellular accumulation of 111 evolutionarily conserved human uORFps and identified a conserved class of human uORFs that encode intrinsic barriers to productive translation and provide a rigorous framework for understanding how noncanonical coding sequences shape the human proteome.
Hinata Hashimoto, Taichi Akase, H. Kurasawa et al.· bioRxiv· 0 citations
Engineering mRNA stability is a promising yet underexplored approach for improving recombinant protein production in bacterial systems. In this study, we evaluated the effect of synthetic 3'-UTR hairpin structures on mRNA stability and protein yield in Escherichia coli using two SUMO-fusion expression systems. Hairpin elements with defined structural features were introduced downstream of the coding sequence. In all constructs, 3'-UTR hairpins increased mRNA half-life, with stabilization ranging from approximately 2-fold to 3-fold (n = 3 biological replicates). In the SUMO-SARS-CoV-2-derived peptide system, enhanced transcript stability was accompanied by a marked increase in specific cellular fusion-protein content, reaching up to 6.8-fold relative to the control (n = 3). In the SUMO-liraglutide-derived peptide system, mRNA stabilization was also pronounced, and the increase in specific cellular fusion-protein content reached approximately 3-fold (p < 0.001, n = 6). These findings show that 3'-UTR engineering is an effective strategy for modulating mRNA stability in *E. coli*, but the quantitative relationship between transcript persistence and protein accumulation is context-dependent and likely influenced by additional factors, including translation efficiency. Overall, engineering of 3'-terminal RNA structures provides a practical tool for post-transcriptional tuning of recombinant expression systems.
Z. Khasanshina, M. Yarovikova, E. Buslaeva et al.· Protein Expression and Purif...· 0 citations
Riboswitches are structured non-coding RNA elements that regulate gene expression in response to small molecules; they serve as valuable systems in both public health and biophysical research by elucidating principles around RNA–ligand interactions, structure, and cellular function. Traditional approaches to studying riboswitches have relied on low-throughput techniques such as reporter assays or gel electrophoresis analysis of transcriptional products, which are limited in scalability. In this study, we present a high-throughput protocol to characterize the transcriptional activity of nearly 2,000 natural variants of the fluoride riboswitch in in vitro transcription. Starting with bioinformatics, we compiled a comprehensive dataset of riboswitch variants and then employed massive parallel oligonucleotide synthesis to generate an oligo pool of the riboswitch library. This pool was transcribed in vitro, converted into an Illumina-compatible next-generation sequencing (NGS) library, and analyzed to identify transcriptionally active riboswitch candidates. The workflow integrates natural riboswitch bioinformatic acquisition into a quantitative readout in a single streamlined pipeline, enabling large-scale exploration of transcriptional riboswitch function. This protocol offers a scalable method for mapping genotype-to-function relationships across transcriptional riboswitch families, accelerating the identification of functional variants for desired applications. Key features • Bioinformatics to acquire full sequences (aptamer + downstream expression platform) of naturally occurring riboswitches. • E. coli RNA polymerase in vitro transcription to characterize nearly 2,000 fluoride riboswitches and analysis with NGS.
Laura M. Hertz, Julius B. Lucks· Bio-protocol· 0 citations
Genetic code expansion enables the site-specific installation of noncanonical amino acids (ncAAs) into proteins, but its limited efficiency in eukaryotes remains a major barrier to broader application. Here we establish a visual, plug-and-play screening platform to evolve 18S ribosomal DNA in Saccharomyces cerevisiae and identify ribosomal variants that improve ncAA incorporation. The best-performing strain, designated ribo-hyper, increased ncAA-dependent GFP production by 2.9-fold relative to the wild-type rDNA strain and enhanced incorporation across distinct orthogonal aminoacyl-tRNA synthetase/tRNA pairs. Characterization of ribo-hyper showed that global translation activity and cellular growth were moderately reduced. Proteomic analysis further revealed changes in amino acid biosynthesis, translation-related proteins and stress-response pathways, indicating that the engineered ribosome reshapes cellular translation homeostasis. Perturbation of translation quality-control pathways, including the ribosome-rescue factors Dom34 and Hbs1 and the core mRNA exosome component Ski6, reduced ncAA-containing protein output, whereas disruption of ribosome quality-control factor Rqc2 had little effect. These findings support a role for ribosome rescue and associated mRNA turnover in efficient ncAA incorporation in the ribo-hyper strain. Together, our results establish eukaryotic ribosome engineering as a viable strategy for improving genetic code expansion in yeast.
Xiao-Xu Chen, Wentao Shen, Xian-Qing Chen et al.· Synthetic and Systems Biotec...· 0 citations
Programmed -1 ribosomal frameshifting (-1 PRF) is a conserved translational recoding mechanism that expands proteomic diversity and regulates gene expression through RNA structural elements, most notably stimulatory pseudoknots. This mechanism is common in viruses, where it is used to control stoichiometry of viral protein products generated by the host cell to direct viral replication. Despite its biological importance, strategies to selectively modulate frameshifting remain limited. The mammalian retrotransposon-derived gene PEG10 also relies on -1 PRF to produce a fusion protein, gag-pol, which is necessary for reproduction but has also been implicated in neurological diseases. Here, we establish an antisense oligonucleotide (ASO) targeting an RNA structural element as an effective approach to tune PEG10 frameshifting. Using structure prediction, systematic antisense tiling across the PEG10 pseudoknot, and multiple model systems, we identify a discrete functional hotspot within the lower RNA stem that governs frameshift efficiency. ASOs targeting this region selectively suppress gag-pol production with minimal impact on gag, thereby shifting the ratio of protein products in a dose-dependent manner. Mechanistic dissection using RNase H-active and -inactive ASO designs, pre-annealed duplexes, and fluorescence-based subcellular localization supports a predominantly nuclear mode of action in which ASOs engage nascent PEG10 transcripts and bias pseudoknot folding away from the frameshift-competent conformation. Functional effects are conserved between human cell lines and murine models, including neurons, highlighting the generality of this strategy. Together, our results define RNA structural dynamics as a druggable layer of translational regulation and establish antisense modulation of pseudoknot folding as a way to control endogenous frameshifting. This work provides a conceptual and practical framework for targeting recoding-dependent gene products such as PEG10 in disease and suggests broader applicability of structure-directed ASOs to viral and cellular frameshifting elements.
Ondrej Kostov, Myriam Moreno Swanton, Katie R. Waldon et al.· bioRxiv· 0 citations
We use cookies to run the site and, with your consent, for analytics and to show ads.
See our Cookie Policy.
