Journal: ChemMedChem

Article Title: Integrated target-based and phenotypic screening approaches for the identification of anti-tubercular agents that bind to the mycobacterial adenylating enzyme MbtA

doi: 10.1002/cmdc.201900217

Figure Lengend Snippet: Structures of docked compounds (Sal-AMS and 5) within the M. smegmatis MbtA active site derived from PDB Ref 5KEI: a. and b. Docked conformation of Sal-AMS; c. and d. Docked conformation of compound 5. The ligands are represented as sticks and the protein shown as a surface (a. and c.) or selected residues shown as green sticks (c. and d.). Polar interactions are shown as yellow dotted lines. Figures were constructed using The PyMOL Molecular Graphics System, Version 1.7 Schrödinger, LLC.

Article Snippet: [ 30 ] Genomic DNA from Mycobacterium smegmatis ATCC 700084/mc(2)155 was obtained from the ATCC Global Bioresource Center (cat.nr.

Techniques: Derivative Assay, Construct

Journal: PLoS Genetics

Article Title: Leaderless Transcripts and Small Proteins Are Common Features of the Mycobacterial Translational Landscape

doi: 10.1371/journal.pgen.1005641

Figure Lengend Snippet: Candidate codons were tested in leadered and leaderless contexts for their activity in initiating translation of the adjacent lacZ gene. Putative positive (ATG) and negative (ATC) controls provided reference activities. The level of β-galactosidase expression in M . smegmatis from leaderless transcripts (upper right green bars beginning with an ATG or GTG codon) was similar to that from leadered transcripts (upper left black bars). CTG and TTG candidate codons were ineffective initiators at the leaderless position. Differences in β -galactosidase activities in M . smegmatis leaderless constructs were not due to effects of the +1 nucleotide (indicated by black background) on transcript abundance, as corresponding leadered constructs were not comparably affected.

Article Snippet: The mc 2 155 strain of M . smegmatis was grown in Middlebrook 7H9 broth supplemented with ADC and 0.05% Tween 80 with shaking at 230 RPM at 37°C to an OD 600 of ~1.0.

Techniques: Activity Assay, Expressing, Construct

Journal: PLoS Genetics

Article Title: Leaderless Transcripts and Small Proteins Are Common Features of the Mycobacterial Translational Landscape

doi: 10.1371/journal.pgen.1005641

Figure Lengend Snippet: (A) Libraries of leader sequences were generated using two overlapping oligonucleotides, each with a single randomized codon positioned either at the leaderless position (+1) or the leadered position (+30), in-frame with the zeocin-resistance ( zeo r ) gene. Self-primed heterodimers were inserted between the promoter and the zeo r gene and transformed into E . coli . The library was electroporated into M . smegmatis . Hygromycin selection allowed maintenance of the complete library, while zeocin selection required translation initiation at either one of the randomized codon sites. Following selection in zeocin, plasmids were recovered and the leader regions amplified for Ion-Torrent sequencing. Deep sequencing of amplicon libraries allowed the unbiased identification and estimation of relative efficiency of initiation codons. (B) A Shine-Dalgarno site was omitted to facilitate direct comparison between leaderless and leadered architectures. Read counts were compiled for each of the 64 possible codons at the leaderless position (columns) and leadered position (rows). Heat map indicates read counts of each combinatorial leaderless/leadered codon pair, from 10 0 (blue) through 10 4 (yellow). Only ATG or GTG at the leaderless position were capable of initiating translation of zeo r . At the leadered codon position, no enrichment indicated that translation initiation did not occur at any of the possible codons. A further reduction of the expected stop codons suggested that they prevented read through of leaderless ribosomes into the zeo r ORF. (C) A Shine-Dalgarno sequence enables efficient use of diverse leadered initiation codons. A consensus Shine-Dalgarno (SD) element was placed upstream of the randomized leadered codon position. Zeocin-resistant pools showed a complex pattern of active translation initiation codons at both the leaderless and leadered positions. The presence of a Shine-Dalgarno supported translation initiation activity of ATG and GTG triplets in the leadered position, as well as the less common TTG and ATT triplets.

Article Snippet: The mc 2 155 strain of M . smegmatis was grown in Middlebrook 7H9 broth supplemented with ADC and 0.05% Tween 80 with shaking at 230 RPM at 37°C to an OD 600 of ~1.0.

Techniques: Generated, Transformation Assay, Selection, Amplification, Sequencing, Comparison, Activity Assay

Journal: PLoS Genetics

Article Title: Leaderless Transcripts and Small Proteins Are Common Features of the Mycobacterial Translational Landscape

doi: 10.1371/journal.pgen.1005641

Figure Lengend Snippet: (A) Zeo-seq viability reporter libraries were generated to determine the sequence context preferences for a SD upstream of a leadered initiation codon. Randomized nucleotides were positioned upstream of a leadered initiation codon, and zeocin selection enriched for Shine-Dalgarno-like sequences, indicating that mycobacteria adhere to this canonical translation criterion. (B) Leaderless translation initiation exhibits no sequence preference in the adjacent mRNA. A block of 6 nt was randomized immediately downstream of a leaderless initiation zeocin reporter construct. Sequences in the recovered pools of zeocin-resistant M . smegmatis were not enriched in composition or motifs in this region. The absence of any detectable enrichment in the randomized region for the leaderless pool indicates that there are no nucleotide preferences for efficient leaderless initiation in mycobacteria downstream of the RTG codon.

Article Snippet: The mc 2 155 strain of M . smegmatis was grown in Middlebrook 7H9 broth supplemented with ADC and 0.05% Tween 80 with shaking at 230 RPM at 37°C to an OD 600 of ~1.0.

Techniques: Generated, Sequencing, Selection, Blocking Assay, Construct

Journal: PLoS Genetics

Article Title: Leaderless Transcripts and Small Proteins Are Common Features of the Mycobacterial Translational Landscape

doi: 10.1371/journal.pgen.1005641

Figure Lengend Snippet: (A) M . tuberculosis leaderless transcripts initiate unannotated small protein ORFs that terminate at the start of the annotated gene downstream more often than expected. All small protein ORF stop codons within 100 nucleotides of an annotated gene start are shown relative to that start codon (0 = coupled RTGA overlap). Three structural classes are identified: uORFs (the small ORF terminates upstream of the annotated start), coupled ORFs (linked by an RTGA tetramer), and overlapping ORFs. The y-axis shows the fraction of small ORFs that terminate a specified distance (x-axis) from the annotated start codon of the downstream gene. (B) One example of a coupled small protein in M . tuberculosis and M . smegmatis , upstream of orthologous genes. The primary sequence of the encoded small protein is not conserved, but the leaderless initiation and coupled linkage is maintained.

Article Snippet: The mc 2 155 strain of M . smegmatis was grown in Middlebrook 7H9 broth supplemented with ADC and 0.05% Tween 80 with shaking at 230 RPM at 37°C to an OD 600 of ~1.0.

Techniques: Sequencing

Journal: PLoS Genetics

Article Title: Leaderless Transcripts and Small Proteins Are Common Features of the Mycobacterial Translational Landscape

doi: 10.1371/journal.pgen.1005641

Figure Lengend Snippet: Small ORFs were identified upstream of annotated orthologous genes (A) cysA2 /Msmeg_5788, (B) Rv0485/Msmeg_0932, and (C) nirA /Msmeg_4527, in the M . tuberculosis and M . smegmatis genomes. Schematic representation of loci in M . tuberculosis (above) or M . smegmatis (below) that encode small proteins (yellow) upstream of genes conserved between these species. The deduced amino acid sequence of each small protein is shown, with the conserved amino acids in gray shaded boxes. The genes downstream in black block arrows are putative members of the same mRNA, and the gene designated by the gray arrow upstream is transcriptionally independent, but shown for context. The amino acid identity with the protein encoded by the corresponding M . tuberculosis gene is indicated below the respective M . smegmatis gene. Below are screen shots of RNA-seq and ribosome profiling profiles in M . smegmatis , and annotated gene predictions from two different annotation algorithms JCVI (black) and PATRIC (blue). Small proteins encoded throughout genomes are poorly annotated; note here that pipeline annotation algorithms predicted none of the small proteins, and in some cases predicted longer proteins on the opposite strand for which we see no transcriptional or translational evidence.

Article Snippet: The mc 2 155 strain of M . smegmatis was grown in Middlebrook 7H9 broth supplemented with ADC and 0.05% Tween 80 with shaking at 230 RPM at 37°C to an OD 600 of ~1.0.

Techniques: Sequencing, Blocking Assay, RNA Sequencing