Machine learning guided batched design of a bacterial Ribosome Binding Site

Optimisation of gene expression levels is an essential part of the organism design process. Fine control of this process can be achieved through engineering transcription and translation control elements, including the ribosome binding site (RBS). Unfortunately, design of specific genetic parts can still be challenging due to lack of reliable design methods. To address this problem, we have created a machine learning guided Design-Build-Test-Learn (DBTL) cycle for the experimental design of bacterial RBSs to show how small genetic parts can be reliably designed using relatively small, high-quality data sets. We used Gaussian Process Regression for the Learn phase of cycle and the Upper Confidence Bound multi-armed bandit algorithm for the Design of genetic variants to be tested in vivo. We have integrated these machine learning algorithms with laboratory automation and high-throughput processes for reliable data generation. Notably, by Testing a total of 450 RBS variants in four DBTL cycles, we experimentally validated RBSs with high translation initiation rates equalling or exceeding our benchmark RBS by up to 34%. Overall, our results show that machine learning is a powerful tool for designing RBSs, and they pave the way towards more complicated genetic devices.

Progressive polysomal association of Upf1, Upf2, and Upf3 as ribosomes traverse mRNA coding regions

SUMMARYUpf1, Upf2, and Upf3 are the central regulators of nonsense-mediated mRNA decay (NMD), the eukaryotic mRNA quality control pathway generally triggered when a premature termination codon is recognized by the ribosome. The NMD-related functions of the Upf proteins likely commence while these factors are ribosome-associated, but little is known of the timing of their ribosome binding, their specificity for ribosomes translating NMD substrates, or the nature and role of any ribosome:Upf complexes. Here, we have elucidated details of the ribosome-associated steps of NMD. By combining yeast genetics with selective ribosome profiling and co-sedimentation analyses of polysomes with wild-type and mutant Upf proteins, our approaches have identified distinct states of ribosome:Upf association. All three Upf factors manifest progressive polysome association as mRNA translation proceeds, but these events appear to be preceded by formation of a Upf1:80S complex as mRNAs initiate translation. This complex is likely executing an early mRNA surveillance function.

RNase III-mediated processing of a trans-acting bacterial sRNA and its cis-encoded antagonist

Bacterial small RNAs (sRNAs) are important post-transcriptional regulators in stress responses and virulence. They can be derived from an expanding list of genomic contexts, such as processing from parental transcripts by RNase E. The role of RNase III in sRNA biogenesis is less well understood despite its well-known roles in rRNA processing, RNA decay, and cleavage of sRNA-mRNA duplexes. Here, we show that RNase III processes a pair of cis-encoded sRNAs (CJnc190 and CJnc180) of the foodborne pathogen Campylobacter jejuni. While CJnc180 processing by RNase III requires CJnc190, In contrast, RNase III processes CJnc190 independent of CJnc180 via cleavage of an intramolecular duplex. We also show that CJnc190 directly represses translation of the colonization factor PtmG by targeting a G-rich ribosome binding site, and uncover that CJnc180 is a cis-acting antagonist of CJnc190, indirectly affecting ptmG regulation. Our study highlights a role for RNase III in sRNA biogenesis and adds cis-encoded RNAs to the expanding diversity of transcripts that antagonize bacterial sRNAs.

Regulation of ER Composition and Extent, and Putative Action in Protein Networks by ER/NE Protein TMEM147

Nuclear envelope (NE) and endoplasmic reticulum (ER) collaborate to control a multitude of nuclear and cytoplasmic actions. In this context, the transmembrane protein TMEM147 localizes to both NE and ER, and through direct and indirect interactions regulates processes as varied as production and transport of multipass membrane proteins, neuronal signaling, nuclear-shape, lamina and chromatin dynamics and cholesterol synthesis. Aiming to delineate the emerging multifunctionality of TMEM147 more comprehensively, we set as objectives, first, to assess potentially more fundamental effects of TMEM147 on the ER and, second, to identify significantly TMEM147-associated cell-wide protein networks and pathways. Quantifying curved and flat ER markers RTN4 and CLIMP63/CKAP4, respectively, we found that TMEM147 silencing causes area and intensity increases for both RTN4 and CLIMP63, and the ER in general, with a profound shift toward flat areas, concurrent with reduction in DNA condensation. Protein network and pathway analyses based on comprehensive compilation of TMEM147 interactors, targets and co-factors then served to manifest novel and established roles for TMEM147. Thus, algorithmically simplified significant pathways reflect TMEM147 function in ribosome binding, oxidoreductase activity, G protein-coupled receptor activity and transmembrane transport, while analysis of protein factors and networks identifies hub proteins and corresponding pathways as potential targets of TMEM147 action and of future functional studies.

Ribosome protection as a mechanism of lincosamide resistance in Mycobacterium abscessus

Mycobacterium abscessus has emerged as a successful pathogen owing to its intrinsic drug resistance. Macrolide and lincosamide antibiotics share overlapping binding sites within the ribosome and common resistance pathways. Nevertheless, while M. abscessus is initially susceptible to macrolides, they are completely resistant to the lincosamide antibiotics. Here we have used RNA sequencing to determine the changes in genes expression in M. abscessus upon exposure to the lincosamide, clindamycin (CLY). We show that Mab_1846 encoding a putative ARE-ABCF protein, was upregulated upon exposure to macrolides and lincosamides, but conferred resistance to CLY alone. An M. smegmatis homologue of Mab_1846 , Ms_5102 , was similarly found to be required for CLY resistance in M. smegmatis . We demonstrate that Ms5102 mediates CLY resistance by directly interacting with the ribosomes and protecting it from CLY inhibition. Additional biochemical characterization showed that ribosome binding is not nucleotide dependent, but ATP hydrolysis is required for dissociation of Ms5102 from the ribosome, as well as for its ability to confer CLY resistance. Finally, we show that in comparison to the macrolides, CLY is a potent inducer of Mab_1846 and the whiB7 regulon, such that exposure of M. abscessus to very low antibiotic concentrations induces a heightened expression of erm41, hflX and Mab_1846 which likely function together to result in a particularly antibiotic resistant state.

ΔGunfold leaderless, a package for high-throughput analysis of translation initiation regions (TIRs) at the transcriptome scale and for leaderless mRNA optimization

Translation initiation is an essential step for fidelity of gene expression, in which the ribosome must bind to the translation initiation region (TIR) and position the initiator tRNA in the P-site (1). For this to occur correctly, the TIR encompassing the ribosome binding site (RBS) needs to be highly accessible (2-5). ΔGunfold is a metric for computing accessibility of the TIR, but there is no automated way to compute it manually with existing software/tools limiting throughput. ΔGunfold leaderless allows users to automate the ΔGunfold calculation to perform high-throughput analysis. Importantly, ΔGunfold leaderless allows calculation of TIRs of both leadered mRNAs and leaderless mRNAs which lack a 5' UTR and which are abundant in bacterial, archaeal, and mitochondrial transcriptomes (4, 6, 7). The ability to analyze leaderless mRNAs also allows one additional feature where users can computationally optimize leaderless mRNA TIRs to maximize their gene expression (8, 9). The ΔGunfold leaderless package facilitates high-throughput calculations of TIR accessibility, is designed to calculate TIR accessibility for leadered and leaderless mRNA TIRs which are abundant in bacterial/archaeal/organellar transcriptomes and allows optimization of leaderless mRNA TIRs for biotechnology.

Engineering a dynamic, controllable infectivity switch in bacteriophage T7

Transcriptional repressors play an important role in regulating phage genomes. Here, we examined how synthetic regulation based on repressors can be to create a dynamic, controllable infectivity switch in bacteriophage T7. We engineered T7 by replacing a large region of the early phage genome with combinations of ligand-responsive promoters and ribosome binding sites (RBS) designed to control the phage RNA polymerase. Phages with the engineered switch showed virulence comparable to wildtype when not repressed, indicating the phage can be engineered without a loss of fitness. When repressed, the most effective switch used a TetR promoter and a weak RBS, resulting in a two-fold increase in latent period (time to lyse host) and change in phage titer. Further, phage activity could be tuned by varying inducer concentrations. Our study provides a proof of concept for a simple circuit for user control over phage infectivity.

Overlapping regions of Caf20 mediate its interactions with the mRNA-5′cap-binding protein eIF4E and with ribosomes

By interacting with the mRNA 5′ cap, the translation initiation factor eIF4E plays a critical role in selecting mRNAs for protein synthesis in eukaryotic cells. Caf20 is a member of the family of proteins found across eukaryotes termed 4E-BPs, which compete with eIF4G for interaction with eIF4E. Caf20 independently interacts with ribosomes. Thus, Caf20 modulates the mRNA selection process via poorly understood mechanisms. Here we performed unbiased mutagenesis across Caf20 to characterise which regions of Caf20 are important for interaction with eIF4E and with ribosomes. Caf20 binding to eIF4E is entirely dependent on a canonical motif shared with other 4E-BPs. However, binding to ribosomes is weakened by mutations throughout the protein, suggesting an extended binding interface that partially overlaps with the eIF4E-interaction region. By using chemical crosslinking, we identify a potential ribosome interaction region on the ribosome surface that spans both small and large subunits and is close to a known interaction site of eIF3. The function of ribosome binding by Caf20 remains unclear.