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Long-range mRNA folding shapes expression and sequence of bacterial genes.

Bacterial gene expression is strongly influenced by local mRNA secondary structure, yet the impact of long-range folding remains poorly understood. Here, we show that sequences hundreds of nucleotides from the mRNA 5' end can act as potent repressors of gene expression through long-range base pairing to the ribosome binding site (RBS), subjecting anti-RBS sequences to negative selection. Using massively parallel reporter assays in Bacillus subtilis, we identify anti-RBS sequences as among the strongest determinants of reduced mRNA abundance across the transcript body. We demonstrate that distal anti-RBS elements engage in long-range folding with the Shine-Dalgarno sequence, blocking ribosome entry and promoting mRNA decay. Consistent with these repressive effects, anti-RBS-like sequences are depleted throughout diverse bacterial coding sequences but not from leaderless transcripts, and introducing distal anti-RBS to native genes reduces expression. Our findings establish that long-range mRNA folding is a conserved force shaping gene expression and constrains coding sequence evolution.

Bacillus subtilis

Rat insulin genes: construction of plasmids containing the coding sequences.

Recombinant bacterial plasmids have been constructed that contain complementary DNA prepared from rat islets of Langerhans messenger RNA. Three plasmids contain cloned sequences representing the complete coding region of rat proinsulin I, part of the preproinsulin I prepeptide, and the untranslated 3' terminal region of the mRNA. A fourth plasmid contains sequences derived from the A chain region of rat preproinsulin II.

Animals

Phenotypic expression in E. coli of a DNA sequence coding for mouse dihydrofolate reductase.

The construction and analysis of bacterial plasmids that contain and phenotypically express a mammalian genetic sequence are described. Such plasmids specify a protein that has enzymatic properties, immunological reactivity and molecular size characteristic of the mouse dihydrofolate reductase, and render host cells resistant to the antimetabolic drug trimethoprim.

Animals

Structure of yeast phenylalanine-tRNA genes: an intervening DNA segment within the region coding for the tRNA.

Sixteen bacterial clones containing sequences complementary to yeast PhetRNA were isolated from a collection of hybrid plasmids containing BamHI restriction endonuclease-generated yeast DNA fragments inserted in the plasmid vector pBR315. Ten of these clones contained hybrid plasmids with distinct BamHI fragments. The sequence of the Phe-tRNA structural genes and adjacent regions of three of these clones is reported here. In the region flanking the tRNA gene, the sequence of two of the cloned DNAs is similar; the sequence of the third varies considerably. All three of the tRNA genes are bordered by A,T-rich regions. In particular, near the region coding for the 3' end of the tRNA there is a long sequence of As in the coding strand. This is reminiscent of the region of termination of transcription of the yeast 5S rRNA gene. The sequences coding for the Phe-tRNA contain an additional segment of 18 or 19 base pairs (depending upon the clone) not predicted by the yeast Phe-tRNA sequence. These intervening segments are nearly identical in the three clones and are located within the structural gene, two base pairs from the nucleotides coding for the tRNA anticodon.

Anticodon

Sequence of the 3'-noncoding and adjacent coding regions of human gamma-globin mRNA.

In cloning human fetal globin cDNA in bacterial plasmids, we obtained a recombinant which contained a fragment of gammg-globin cDNA corresponding to the region from amino acid 99 to the poly A. We determined a sequence of 169 nucleotides which included the complete 3' non-coding region of the gamma-globin mRNA. The codon for amino acid 136 was GCA, indicating that this cloned fragment was derived from the Agamma-globin gene. In conjunction with the surrounding sequences, the GCA codon provides the Agamma-species with a unique CTGCAG hexanucleotide that is recognized by the restriction enzyme Pst I. The 3'-untranslated region of the gamma-globin mRNA consists of 90 nucleotides, and shares little homology with that of the human beta-globin mRNA. As in other mammalian mRNAs, a symmetrical sequence and the hexanucleotide AAUAAA are present.

Base Sequence

Induction of base-pair substitution and frameshift mutations in wild-type and repair-deficient strains of Salmonella typhimurium by the photodynamic action of methylene blue.

Induction of back mutations to prototrophy by methylene blue (MB)-sensitized photodynamic (PD) treatment has been studied in wild-type and repair-deficient strains of Salmonella typhimurium carrying either the base-pair substitution mutation hisG46 or the frameshift mutation hisD30529 We found that reversion of the hisG46 mutation was increased in a strain carrying a uvrB deletion and decreased in a strain carrying a recA-type mutation. Reversion of the hisD3052 (frameshift) mutation, on the other hand, was decreased in both uvrB deletion and recA-type strains. The former results are consistent with the hypothesis that the majority of MB-sensitized PD-INDUCED BASE-PAIR SUBSTITUTION MUTATIONS ARIse by a mechanism similar to that currently believed to be involved in UV mutagenesis. The latter results suggest that PD-induced frameshift mutations may arise in some other way, and two possible mechanisms involving sequential action of the excision repair and recombinational repair pathways are considered.

Base Sequence

The predicted secondary structure of the N-terminal sequence of the lac repressor and proposed models for its complexation to the lac operator.

Rules for the prediction of protein conformation (Chou, P. Y., and Fasman, G. D. (1974), Biochemistry 13, 211, 222) have been applied to the N-terminal sequence 1-60 of the lac repressor. This analysis predicts beta structure at sequences 4-9 and 15-20, helices at 26-32, 38-45, and 52-57, and beta turns at 48-51 and 14-17. Repressor mutants lacking operator binding capacity in which Pro replaces Ser-16 and Ala replaces Thr-19 (Weber, K., Platt, T., Ganem, D., and Miller, J. H. (1972), Proc. Natl. Acad. Sci. U.S.A. 69, 3624) have no effect on the prediction of beta structure at residues 15 to 20, which suggests that the polar side chains of Ser-16, Tyr-17, Gln-18, and Thr-19 participate in intermolecular hydrogen bonding with conplementary polar groups on the lac operator. The loss of operator binding capacity on replacement of Ala by Val at position 53 in the repressor results from a predicted secondary structural change from helix to beta structure for residues 52-57 which can be transmitted to the N-terminal sequence via a beta turn at residues 48-51. The basic residues at positions 33, 35, and 37 between the helical regions 26-32 and 38-45 probably bind to the phosphate groups on the operator on complexation. It is proposed that complex formation involves the interaction of either a beta structure (residues 15-20) or a right-hand twisted antiparallel beta-pleated sheet (residues 4-9 and 12-20) with operator DNA.

Amino Acid Sequence

Cellular function of the GndA microprotein during heat shock.

Over the past 15 years, hundreds of previously undiscovered bacterial small open reading frames (sORFs) encoding microproteins of fewer than fifty amino acids have been identified. Biological functions have been ascribed to an increasing number of microproteins from intergenic regions and small RNAs, and many play integral roles in bacterial stress responses. However, despite numbering in the dozens in Escherichia coli, and hundreds in humans, same-strand frameshifted sORFs that internally overlap protein coding sequences remain understudied. To provide insight into nested genes, we characterized GndA, a frameshifted 36-amino acid microprotein nested within the 6-phosphogluconate dehydrogenase (6PGD) coding sequence. Using precise genome editing, we demonstrate independent contributions of GndA and 6PGD to cell growth at high temperature. GndA associates with membrane-associated complexes associated with electron transport and ATP generation, and supports ATP homeostasis during heat shock. Functional characterization of GndA thus adds to the catalog of bacterial microproteins that function in stress responses, while providing clear genetic evidence for the importance of an overlapping gene to cellular fitness.

6PGD

Demixer: a probabilistic generative model to delineate different strains of a microbial species in a mixed infection sample.

MOTIVATION: Multi-drug resistant or hetero-resistant tuberculosis (TB) hinders the successful treatment of TB. Hetero-resistant TB occurs when multiple strains of the TB-causing bacterium with varying degrees of drug susceptibility are present in an individual. Existing studies predicting the proportion and identity of strains in a mixed infection sample rely on a reference database of known strains. A main challenge then is to identify de novo strains not present in the reference database, while quantifying the proportion of known strains. RESULTS: We present Demixer, a probabilistic generative model that uses a combination of reference-based and reference-free techniques to delineate mixed infection strains in whole genome sequencing (WGS) data. Demixer extends a topic model widely used in text mining to represent known mutations and discover novel ones. Parallelization and other heuristics enabled Demixer to process large datasets like CRyPTIC (Comprehensive Resistance Prediction for Tuberculosis: an International Consortium). In both synthetic and experimental benchmark datasets, our proposed method precisely detected the identity (e.g. 91.67% accuracy on the experimental in vitro dataset) as well as the proportions of the mixed strains. In real-world applications, Demixer revealed novel high confidence mixed infections (101 out of 1963 Malawi samples analysed), and new insights into the global frequency of mixed infection (2% at the most stringent threshold in the CRyPTIC dataset) and its significant association to drug resistance. Our approach is generalizable and hence applicable to any bacterial and viral WGS data. AVAILABILITY AND IMPLEMENTATION: All code relevant to Demixer is available at https://github.com/BIRDSgroup/Demixer.

Mycobacterium tuberculosis

Genes and non-coding DNA sequences.

Many new categories of DNA sequence organization in higher organisms have been discovered in the last 20 years. Most of these are not found in bacterial and each in turn is hailed as a likely candidate for those regulatory elements which are assumed to distinguish higher from lower organisms. This article stresses the similarities between their sequence organizations and the continuing lack of convincing function for any of them. It suggests that these sequences often appear after chance recombinational events and their presence then facilitates further change. Within the constraints of proper function the genome is clearly able to find many organizational solutions of apparently equal advantage to the organism. Finally, an example of rapid genome reorganization resulting from selection for resistance to the drug methotrexate is considered.

Animals

Purification and characterization of a new factor which restores protein synthesis in a conditionally lethal mutant of Escherichia coli.

The mutant Escherichia coli strain, N4316, has a temperature-sensitive defect in a protein factor required for translation in vitro of bacteriophage f2 RNA. We have purified the normal counterpart of this factor from a wild-type strain, using as an assay its ability to restore the activity of mutant extracts at non-permissive temperature. Our final preparation is free of known initiation, propagation, and release factors, proving that the factor is a new component required for translation. The new factor has a molecular weight of 95000 with preliminary data suggesting a subunit structure. 70% of this protein is found in the soluble-cell fraction, the rest being associated with 70-S ribosomes. Kinetic analyses indicate that the factor acts early in translation. Expression of the defect is highly dependent on the Mg2+ concentration, no temperature-sensitivity being apparent at 15 mM Mg2+. At lower Mg2+ concentrations, the defect is expressed only with natural mRNAs such as f2 RNA, and not with artifical polymers such as poly(U). This specificity suggests that the factor may function in events coded by special sequences in the natural messengers

Bacterial Proteins

Determinant of cistron specificity in bacterial ribosomes.

The sequence of the 3'-terminus of 16S RNA from different bacteria has been determined. Complementarity relationships between this sequence and a purine-rich tract in the ribosome binding site of different bacterial mRNAs suggest that the 3'-end of 16S RNA determines the intrinsic capacity of ribosomes to translate a particular cistron.

Bacillus

HindII and HindIII restriction maps of the attphi80-tonB-trp region of the Escherichia coli genome, and location of the tonB gene.

The HindII and HindIII restriction maps of the attphi80-tonB-trp region of the Escherichia coli chromosome are presented. Analysis of phage DNAs carrying tonB mutations has allowed identification of a 1,730-base pair HindII fragment containing at least part of the tonB gene. This fragment is 4,020 base pairs from the end of trpA, with the total distance from attphi80 to trpA being 6,550 +/- 800 base pairs. Properties of hybrid plasmids containing insertions of various tonB+ restriction fragments suggest that tonB lies completely within the 1,730-base pair fragment. In addition, apparent fusions of beta-galactoside to proteins within the tonB region suggest that the entire region codes for more than one polypeptide.

Bacterial Proteins

dnaG (primase)-dependent origins of DNA replication. Nucleotide sequences of the negative strand initiation sites of bacteriophages St-1, phi K, and alpha 3.

The simplest known origins of DNA replication occur in the single-stranded bacteriophages. In one set of phages, negative strand synthesis is initiated by a single protein, the product of the Escherichia coli replication gene dnaG. Evidently, in these phages--G4, St-1, phi K, and alpha 3--the origin for negative strand synthesis consists of a nucleic acid element capable of direct recognition by the dnaG priming protein. We have located and sequenced the origins of negative strand synthesis in St-1, phi K, and alpha 3, and compared them with the origin sequence previously determined for G4. In each case, the point at which the negative strand is initiated can be identified at the nucleotide level. The data lead to the following conclusions: 1. In all four phages, the negative strand initiation site occurs within an intercistronic region of approximately 135 bases. While in G4, the origin lies between genes specifying the viral coat proteins F and G, the origin is shifted in St-1, phi K, and alpha 3 to a position between coat protein genes G and H. 2. Extensive nucleotide conservation exists at the negative strand origin, but does not extend into the adjacent coding regions. The conserved origin DNA occurs in two regions, 42 and 45 bases long, which are separated by 13 bases of divergent sequence. 3. Correlated with the two stretches of conserved nucleotide sequence are two regions of potential secondary structure. The start point of negative strand synthesis lies just prior to one of these hairpins. Similarities in both primary sequence and secondary structure can be found between the negative strand origins of G4, St-1, phi K, and alpha 3 and the general origin regions of bacteriophage lambda and of E. coli.

Base Sequence

Nucleotide sequence of the intercistronic region preceding the gene for RNA polymerase subunit alpha in Escherichia coli.

The gene for RNA polymerase subunit alpha is a co-transcribed with several ribosomal protein genes (Jaskunas, S.R., Burgess, R.R., and Nomura, M. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 5036-5040). The DNA sequence whicch codes for the COOH-terminal amino acids of S4 and the NH2-terminal amino acids of alpha, and the 25 nucleotide intercistronic sequence have been determined. This short distance supports the idea that some post-transcriptional regulation determines the differential synthesis of alpha and ribosomal proteins.

Amino Acid Sequence

Landscape of retron diversity across the SPIRE microbial metagenome resource reveals candidate novel type XI-like lineages.

Retrons are bacterial genetic elements encoding a specialized reverse transcriptase (RT) that synthesizes multicopy single-stranded DNA and are increasingly recognized as components of bacterial anti-phage defense systems. However, their diversity and ecological distribution across large-scale genomic resources remain poorly characterized. Here, we surveyed retron RTs across the SPIRE representative metagenome collection, a non-redundant, species-level data set spanning diverse microbial habitats. Using a curated panel of type-specific hidden Markov models, we identified retrons representing all canonical types together with additional divergent lineages. Retron distribution showed strong taxonomic and ecological structuring, with some groups restricted to specific bacterial phyla, whereas others were broadly distributed across environmental categories. Systematic novelty assessment identified two candidate type XI-like lineages, TXI_C2like and TXI_noncan_h, characterized by protease-independent architectures and distinct accessory modules associated with WYL- and DnaB_C-containing proteins, respectively. De novo covariance-based analyses further identified candidate msr/msd-like non-coding RNA structures in both lineages, supporting conservation of the canonical RT-ncRNA organizational framework despite extensive sequence divergence. Together, these findings expand the known diversity of retron systems and identify type XI-like retrons as a dynamic and previously underexplored evolutionary group.IMPORTANCERetrons are bacterial genetic elements that are increasingly exploited as programmable tools for genome editing, molecular recording, and biosensing in addition to their natural role in anti-phage defense. Despite this growing biotechnological interest, the true diversity of retrons across the bacterial world has remained largely unmapped. By mining a resource of over 100,000 processed microbial metagenomes, we uncovered thousands of retron sequences spanning known types as well as previously unrecognized lineages and found that their distribution is strongly shaped by both bacterial taxonomy and ecological niche. Among these, we identified two candidate new lineages related to type XI retrons that lack the protease domain typical of this group but instead carry distinct accessory proteins, expanding the known architectural diversity of these systems. These findings broaden the catalog of retron diversity available for functional characterization and biotechnological engineering and provide a framework for prioritizing candidate lineages for future experimental validation.

effectors