Conservation of Transcription Start Sites within Genes across a Bacterial Genus

ABSTRACT Transcription start sites (TSSs) lying inside annotated genes, on the same or opposite strand, have been observed in diverse bacteria, but the function of these unexpected transcripts is unclear. Here, we use the metal-reducing bacterium Shewanella oneidensis MR-1 and its relatives to study the evolutionary conservation of unexpected TSSs. Using high-resolution tiling microarrays and 5′-end RNA sequencing, we identified 2,531 TSSs in S. oneidensis MR-1, of which 18% were located inside coding sequences (CDSs). Comparative transcriptome analysis with seven additional Shewanella species revealed that the majority (76%) of the TSSs within the upstream regions of annotated genes (gTSSs) were conserved. Thirty percent of the TSSs that were inside genes and on the sense strand (iTSSs) were also conserved. Sequence analysis around these iTSSs showed conserved promoter motifs, suggesting that many iTSS are under purifying selection. Furthermore, conserved iTSSs are enriched for regulatory motifs, suggesting that they are regulated, and they tend to eliminate polar effects, which confirms that they are functional. In contrast, the transcription of antisense TSSs located inside CDSs (aTSSs) was significantly less likely to be conserved (22%). However, aTSSs whose transcription was conserved often have conserved promoter motifs and drive the expression of nearby genes. Overall, our findings demonstrate that some internal TSSs are conserved and drive protein expression despite their unusual locations, but the majority are not conserved and may reflect noisy initiation of transcription rather than a biological function. IMPORTANCE The first step of gene expression is the initiation of transcription from promoters, which have been traditionally thought to be located upstream of genes. Recently, studies showed that in diverse bacteria, promoters are often located inside genes. It has not been clear if these unexpected promoters are important to the organism or if they result from transcriptional noise. Here, we identify and examine promoters in eight related bacterial species. Promoters that lie within genes on the sense strand are often conserved as locations and in their sequences. Furthermore, these promoters often affect the bacterium’s growth. Thus, many of these unexpected promoters are likely functional. Fewer promoters that lie within genes on the antisense strand are conserved, but the conserved ones seem to drive the expression of nearby genes.

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Structural organization of upstream exons and distribution of transcription start sites in the chicken c-myb gene

Molecular and Cellular Biology ◽

10.1128/mcb.9.2.837-843.1989 ◽

1989 ◽

Vol 9 (2) ◽

pp. 837-843

Author(s):

S L Hahn ◽

M Hahn ◽

W S Hayward

Keyword(s):

Binding Sites ◽

Noncoding Region ◽

Open Reading Frames ◽

Transcription Start ◽

S1 Nuclease ◽

Transcription Start Sites ◽

Conserved Sequence ◽

Myb Gene ◽

Putative Transcription Factor ◽

Myb Genes

We mapped and sequenced three upstream exons of the chicken c-myb gene and the regions flanking the first coding exon. We found multiple potential binding sites for transcription factors in the 5'-noncoding region, a T-rich stretch of 78 base pairs (bp) (68% T) in the first intron, and four fairly long open reading frames in the antisense direction of the first coding exon and its flanking regions. Three major transcription start sites, contained within a single 11-bp region, were identified by S1 nuclease analysis and primer extension. A sequence comparison of the avian and murine c-myb genes revealed a highly conserved sequence of 124 bp in the 5'-noncoding region. Its location between the putative transcription factor binding sites and the major transcription start sites suggests that it may play an important regulatory role in c-myb expression.

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Self-assembling short immunostimulatory duplex RNAs with broad spectrum antiviral activity

10.1101/2021.11.19.469183 ◽

2021 ◽

Author(s):

Longlong Si ◽

Haiqing Bai ◽

Crystal Yuri Oh ◽

Tian Zhang ◽

Amanda Jiang ◽

...

Keyword(s):

Broad Spectrum ◽

Influenza A ◽

Poly I:C ◽

Antisense Strand ◽

Sequence Motif ◽

Type I ◽

Conserved Sequence ◽

Self Assembling ◽

Sense Strand ◽

Wide Range

The current COVID-19 pandemic highlights the need for broad-spectrum antiviral therapeutics. Here we describe a new class of self-assembling immunostimulatory short duplex RNAs that potently induce production of type I and type III interferon (IFN-I and IFN-III), in a wide range of human cell types. These RNAs require a minimum of 20 base pairs, lack any sequence or structural characteristics of known immunostimulatory RNAs, and instead require a unique conserved sequence motif (sense strand: 5'-C, antisense strand: 3'-GGG) that mediates end-to-end dimer self-assembly of these RNAs by Hoogsteen G-G base-pairing. The presence of terminal hydroxyl or monophosphate groups, blunt or overhanging ends, or terminal RNA or DNA bases did not affect their ability to induce IFN. Unlike previously described immunostimulatory siRNAs, their activity is independent of TLR7/8, but requires the RIG-I/IRF3 pathway that induces a more restricted antiviral response with a lower proinflammatory signature compared with poly(I:C). Immune stimulation mediated by these duplex RNAs results in broad spectrum inhibition of infections by many respiratory viruses with pandemic potential, including SARS-CoV-2, SARS-CoV, MERS-CoV, and influenza A, as well as the common cold virus HCoV-NL63 in both cell lines and human Lung Chips that mimic organ-level lung pathophysiology. These short dsRNAs can be manufactured easily, and thus potentially could be harnessed to produce broad-spectrum antiviral therapeutics at low cost.

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Transcriptional and post-transcriptional regulation of young genes in plants

10.1101/2021.12.20.473517 ◽

2021 ◽

Author(s):

Vivek Kumar Raxwal ◽

Somya Singh ◽

Manu Agarwal ◽

Karel Riha

Keyword(s):

Transcriptional Regulation ◽

Purifying Selection ◽

Chromatin Conformation ◽

Transcription Start ◽

Transcription Start Sites ◽

New Genes ◽

Conditional Expression ◽

Nonsense Mediated Rna Decay ◽

Post Transcriptional Regulation ◽

Low Expression

New genes continuously emerge from non-coding DNA or by diverging from existing genes, but most of them are rapidly lost and only a few become fixed within the population. We hypothesized that young genes are subject to transcriptional and post-transcriptional regulation to limit their expression and minimize their exposure to purifying selection. We found that young genes in rice have relatively low expression levels, which can be attributed to distal enhancers, and closed chromatin conformation at their transcription start sites (TSS). The chromatin in TSS regions can be re-modeled in response to abiotic stress, indicating conditional expression of young genes. Furthermore, transcripts of young genes in Arabidopsis tend to be targeted by nonsense-mediated RNA decay, presenting another layer of regulation limiting their expression. Together, these data suggest that transcriptional and post-transcriptional mechanisms contribute to the conditional expression of young genes, which may alleviate purging selection while providing an opportunity for phenotypic exposure and functionalization.

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The TetR-Type Transcriptional Repressor RolR from Corynebacterium glutamicum Regulates Resorcinol Catabolism by Binding to a Unique Operator,rolO

Applied and Environmental Microbiology ◽

10.1128/aem.01304-12 ◽

2012 ◽

Vol 78 (17) ◽

pp. 6009-6016 ◽

Cited By ~ 11

Author(s):

Tang Li ◽

Kexin Zhao ◽

Yan Huang ◽

Defeng Li ◽

Cheng-Ying Jiang ◽

...

Keyword(s):

Corynebacterium Glutamicum ◽

Promoter Activity ◽

Activity Analysis ◽

Inverted Repeats ◽

Catabolic Pathway ◽

Transcription Start ◽

Content Type ◽

Transcription Start Sites ◽

Reverse Transcription Pcr ◽

Promoter Activity Analysis

ABSTRACTTherol(designated forresorcinol) gene clusterrolRHMDis involved in resorcinol catabolism inCorynebacterium glutamicum, and RolR is the TetR-type regulator. In this study, we investigated how RolR regulated the transcription of therolgenes inC. glutamicum. The transcription start sites and promoters ofrolRandrolHMDwere identified. Quantitative reverse transcription-PCR and promoter activity analysis indicated that RolR negatively regulated the transcription ofrolHMDand of its own gene. Further, a 29-bp operatorrolOwas located at the intergenic region ofrolRandrolHMDand was identified as the sole binding site for RolR. It contained two overlapping inverted repeats and they were essential for RolR-binding. The binding of RolR torolOwas affected by resorcinol and hydroxyquinol, which are the starting compounds of resorcinol catabolic pathway. These two compounds were able to dissociate RolR-rolOcomplex, thus releasing RolR from the complex and derepressing the transcription ofrolgenes inC. glutamicum. It is proposed that the binding of RolR to its operatorrolOblocks the transcription ofrolHMDand of its own gene, thus negatively regulated resorcinol degradation inC. glutamicum.

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Widespread divergent transcription from prokaryotic promoters

10.1101/2020.01.31.928960 ◽

2020 ◽

Cited By ~ 1

Author(s):

Emily Warman ◽

David Forrest ◽

Joseph T. Wade ◽

David C. Grainger

Keyword(s):

Dna Sequences ◽

Transcription Start ◽

Promoter Elements ◽

Early Step ◽

Transcription Start Sites ◽

Bidirectional Promoters ◽

Initiation Of Transcription ◽

Dna Strands ◽

Divergent Transcription

ABSTRACTPromoters are DNA sequences that stimulate the initiation of transcription. In all prokaryotes, promoters are believed to drive transcription in a single direction. Here we show that prokaryotic promoters are frequently bidirectional and drive divergent transcription. Mechanistically, this occurs because key promoter elements have inherent symmetry and often coincide on opposite DNA strands. Reciprocal stimulation between divergent transcription start sites also contributes. Horizontally acquired DNA is enriched for bidirectional promoters suggesting that they represent an early step in prokaryotic promoter evolution.

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Phenotypic Parallelism during Experimental Adaptation of a Free-Living Bacterium to the Zebrafish Gut

mBio ◽

10.1128/mbio.01519-20 ◽

2020 ◽

Vol 11 (4) ◽

Author(s):

Jarrett F. Lebov ◽

Brandon H. Schlomann ◽

Catherine D. Robinson ◽

Brendan J. M. Bohannan

Keyword(s):

Experimental Evolution ◽

Bacterial Species ◽

Shewanella Oneidensis ◽

Free Living ◽

Content Type ◽

Larval Zebrafish ◽

Health And Development ◽

Living Bacterium ◽

Animal Hosts ◽

Free Living Bacterium

ABSTRACT Although animals encounter a plethora of bacterial species throughout their lives, only a subset colonize vertebrate digestive tracts, and these bacteria can profoundly influence the health and development of their animal hosts. However, our understanding of how bacteria initiate symbioses with animal hosts remains underexplored, and this process is central to the assembly and function of gut bacterial communities. Therefore, we used experimental evolution to study a free-living bacterium as it adapts to a novel vertebrate host by serially passaging replicate populations of Shewanella oneidensis through the intestines of larval zebrafish (Danio rerio). After approximately 200 bacterial generations, isolates from evolved populations improved their ability to colonize larval zebrafish during competition against their unpassaged ancestor. Genome sequencing revealed unique sets of mutations in the two evolved isolates exhibiting the highest mean competitive fitness. One isolate exhibited increased swimming motility and decreased biofilm formation compared to the ancestor, and we identified a missense mutation in the mannose-sensitive hemagglutinin pilus operon that is sufficient to increase fitness and reproduce these phenotypes. The second isolate exhibited enhanced swimming motility but unchanged biofilm formation, and here the genetic basis for adaptation is less clear. These parallel enhancements in motility and fitness resemble the behavior of a closely related Shewanella strain previously isolated from larval zebrafish and suggest phenotypic convergence with this isolate. Our results demonstrate that adaptation to the zebrafish gut is complex, with multiple evolutionary pathways capable of improving colonization, but that motility plays an important role during the onset of host association. IMPORTANCE Although animals encounter many bacterial species throughout their lives, only a subset colonize vertebrate digestive tracts, and these bacteria can profoundly influence the health and development of their animal hosts. We used experimental evolution to study a free-living bacterium as it adapts to a novel vertebrate host by serially passaging replicate populations of Shewanella oneidensis through the intestines of larval zebrafish (Danio rerio). Our results demonstrate that adaptation to the zebrafish gut is complex, with multiple evolutionary pathways capable of improving colonization, but that motility plays an important role during the onset of host association.

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Where To Begin? Mapping Transcription Start Sites Genome-Wide in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.02410-14 ◽

2014 ◽

Vol 197 (1) ◽

pp. 4-6 ◽

Cited By ~ 6

Author(s):

Joseph T. Wade

Keyword(s):

Escherichia Coli ◽

Transcription Start ◽

Content Type ◽

Transcription Start Sites ◽

Extended Region of Nodulation Genes in Rhizobium meliloti 1021. II. Nucleotide Sequence, Transcription Start Sites and Protein Products

Genetics ◽

10.1093/genetics/117.2.191 ◽

1987 ◽

Vol 117 (2) ◽

pp. 191-201

Author(s):

Robert F Fisher ◽

Jean A Swanson ◽

John T Mulligan ◽

Sharon R Long

Keyword(s):

Binding Site ◽

Rhizobium Meliloti ◽

Open Reading Frames ◽

Translation System ◽

Transcription Start ◽

Nod Genes ◽

Transcription Start Sites ◽

Conserved Sequence ◽

Reading Frames

ABSTRACT We have established the DNA sequence and analyzed the transcription and translation products of a series of putative nodulation (nod) genes in Rhizobium meliloti strain 1021. Four loci have been designated nodF, nodE, nodG and nodH. The correlation of transposon insertion positions with phenotypes and open reading frames was confirmed by sequencing the insertion junctions of the transposons. The protein products of these nod genes were visualized by in vitro expression of cloned DNA segments in a R. meliloti transcription-translation system. In addition, the sequence for nodG was substantiated by creating translational fusions in all three reading frames at several points in the sequence; the resulting fusions were expressed in vitro in both E. coli and R. meliloti transcription-translation systems. A DNA segment bearing several open reading frames downstream of nodG corresponds to the putative nod gene mutated in strain nod-216. The transcription start sites of nodF and nodH were mapped by primer extension of RNA from cells induced with the plant flavone, luteolin. Initiation of transcription occurs approximately 25 bp downstream from the conserved sequence designated the "nod box," suggesting that this conserved sequence acts as an upstream regulator of inducible nod gene expression. Its distance from the transcription start site is more suggestive of an activator binding site rather than an RNA polymerase binding site.

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Functional Specificity of Extracellular Nucleases of Shewanella oneidensis MR-1

Applied and Environmental Microbiology ◽

10.1128/aem.07895-11 ◽

2012 ◽

Vol 78 (12) ◽

pp. 4400-4411 ◽

Cited By ~ 14

Author(s):

Magnus Heun ◽

Lucas Binnenkade ◽

Maximilian Kreienbaum ◽

Kai M. Thormann

Keyword(s):

Biofilm Formation ◽

Bacterial Species ◽

Shewanella Oneidensis ◽

Large Cell ◽

Sole Source ◽

Extracellular Dna ◽

Divalent Metal Ions ◽

Content Type ◽

Highly Active ◽

Extracellular Nuclease

ABSTRACTBacterial species such asShewanella oneidensisMR-1 require extracellular nucleolytic activity for the utilization of extracellular DNA (eDNA) as a source of nutrients and for the turnover of eDNA as a structural matrix component during biofilm formation. We have previously characterized two extracellular nucleases ofS. oneidensisMR-1, ExeM and ExeS. Although both are involved in biofilm formation, they are not specifically required for the utilization of eDNA as a nutrient. Here we identified and characterized EndA, a third extracellular nuclease ofShewanella. The heterologously overproduced and purified protein was highly active and rapidly degraded linear and supercoiled DNAs of various origins. Divalent metal ions (Mg2+or Mn2+) were required for function.endAis cotranscribed withphoA, an extracellular phosphatase, and is not upregulated upon phosphostarvation. Deletion ofendAabolished both extracellular degradation of DNA byS. oneidensisMR-1 and the ability to use eDNA as a sole source of phosphorus. PhoA is not strictly required for the exploitation of eDNA as a nutrient. The activity of EndA prevents the formation of large cell aggregates during planktonic growth. However, in contrast to the findings for ExeM,endAdeletion had only minor effects on biofilm formation. The findings strongly suggest that the extracellular nucleases ofS. oneidensisexert specific functions required under different conditions.

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