Characterization of in vitro transcription initiation and termination sites in Col E1 DNA

Complete cDNA copies of each of the brome mosaic virus genomic RNAs (3.2, 2.8, and 2.1 kilobases in length) were cloned in a novel transcription vector, pPM1, designed to provide exact control of the transcription initiation site. After cleavage at a unique EcoRI site immediately downstream of the inserted cDNA, these clones can be transcribed in vitro by Escherichia coli RNA polymerase to yield complete copies of the brome mosaic virus RNAs. Dideoxy sequencing of 5' transcript cDNA runoff products and direct sequencing of 32P-3'-end-labeled transcripts show that such transcripts initiate at the same 5' position as natural viral RNA and terminate within the EcoRI runoff site after copying the entire viral RNA sequence. When synthesized in the presence of m7GpppG, the transcripts bear the natural capped 5' terminus of brome mosaic virus RNAs. Such transcripts direct the in vitro translation of proteins which coelectrophorese with the translation products of natural brome mosaic virus RNAs. pPM1 should facilitate in vitro production of other viral and nonviral RNAs.

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σIfromBacillus subtilis: Impact on Gene Expression and Characterization of σI-Dependent Transcription That Requires New Types of Promoters with Extended −35 and −10 Elements

Journal of Bacteriology ◽

10.1128/jb.00251-18 ◽

2018 ◽

Vol 200 (17) ◽

Cited By ~ 4

Author(s):

Olga Ramaniuk ◽

Martin Převorovský ◽

Jiří Pospíšil ◽

Dragana Vítovská ◽

Olga Kofroňová ◽

...

Keyword(s):

Gene Expression ◽

Bacillus Subtilis ◽

Iron Metabolism ◽

Transcription Initiation ◽

Iron Homeostasis ◽

Model Organism ◽

Content Type ◽

Σ Factor

ABSTRACTThe σIsigma factor fromBacillus subtilisis a σ factor associated with RNA polymerase (RNAP) that was previously implicated in adaptation of the cell to elevated temperature. Here, we provide a comprehensive characterization of this transcriptional regulator. By transcriptome sequencing (RNA-seq) of wild-type (wt) and σI-null strains at 37°C and 52°C, we identified ∼130 genes affected by the absence of σI. Further analysis revealed that the majority of these genes were affected indirectly by σI. The σIregulon, i.e., the genes directly regulated by σI, consists of 16 genes, of which eight (thedhbandykuoperons) are involved in iron metabolism. The involvement of σIin iron metabolism was confirmed phenotypically. Next, we set up anin vitrotranscription system and defined and experimentally validated the promoter sequence logo that, in addition to −35 and −10 regions, also contains extended −35 and −10 motifs. Thus, σI-dependent promoters are relatively information rich in comparison with most other promoters. In summary, this study supplies information about the least-explored σ factor from the industrially important model organismB. subtilis.IMPORTANCEIn bacteria, σ factors are essential for transcription initiation. Knowledge about their regulons (i.e., genes transcribed from promoters dependent on these σ factors) is the key for understanding how bacteria cope with the changing environment and could be instrumental for biotechnologically motivated rewiring of gene expression. Here, we characterize the σIregulon from the industrially important model Gram-positive bacteriumBacillus subtilis. We reveal that σIaffects expression of ∼130 genes, of which 16 are directly regulated by σI, including genes encoding proteins involved in iron homeostasis. Detailed analysis of promoter elements then identifies unique sequences important for σI-dependent transcription. This study thus provides a comprehensive view on this underexplored component of theB. subtilistranscription machinery.

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Formation of an active transcription complex in the Drosophila melanogaster 5S RNA gene is dependent on an upstream region.

Molecular and Cellular Biology ◽

10.1128/mcb.7.6.2046 ◽

1987 ◽

Vol 7 (6) ◽

pp. 2046-2051 ◽

Cited By ~ 18

Author(s):

A D Garcia ◽

A M O'Connell ◽

S J Sharp

Keyword(s):

Drosophila Melanogaster ◽

Transcription Initiation ◽

Rna Polymerase Iii ◽

In Vitro Transcription ◽

Upstream Region ◽

Nucleotide Position ◽

Wild Type ◽

5S Rna ◽

Cell Extracts

We constructed deletion-substitution and linker-scanning mutations in the 5'-flanking region of the Drosophila melanogaster 5S RNA gene. In vitro transcription of these templates in Drosophila and HeLa cell extracts revealed the presence of an essential control region (-30 region) located between nucleotides -39 and -26 upstream of the transcription initiation site: deletion of sequences upstream of nucleotide position -39 had no detectable effect on the wild-type level of in vitro transcription, whereas mutations extending between positions -39 and 1 resulted in templates with decreased transcriptional levels; specifically, deletion and linker-scanning mutations in the -34 to -26 region (-30 region) resulted in loss of transcription. The -30 region is essential for transcription and therefore forms part of the Drosophila 5S RNA gene transcription promoter. Compared with the activity of the wild-type gene, mutant 5S DNAs exhibited no impairment in the ability to sequester limiting transcription factors in a template exclusion competition assay. While we do not know which transcription factor(s) interacts with the -30 region, the possible involvement of RNA polymerase III at this region is discussed.

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Termination-altering mutations in the second-largest subunit of yeast RNA polymerase III.

Molecular and Cellular Biology ◽

10.1128/mcb.15.3.1467 ◽

1995 ◽

Vol 15 (3) ◽

pp. 1467-1478 ◽

Cited By ~ 27

Author(s):

S A Shaaban ◽

B M Krupp ◽

B D Hall

Keyword(s):

Amino Acid ◽

Rna Polymerase ◽

Transcription Initiation ◽

Transcription Termination ◽

Rna Polymerase Iii ◽

In Vitro Transcription ◽

The Other ◽

Polymerase Iii

In order to identify catalytically important amino acid changes within the second-largest subunit of yeast RNA polymerase III, we mutagenized selected regions of its gene (RET1) and devised in vivo assays for both increased and decreased transcription termination by this enzyme. Using as the reporter gene a mutant SUP4-o tRNA gene that in one case terminates prematurely and in the other case fails to terminate, we screened mutagenized RET1 libraries for reduced and increased transcription termination, respectively. The gain in suppression phenotype was in both cases scored as a reduction in the accumulation of red pigment in yeast strains harboring the ade2-1 ochre mutation. Termination-altering mutations were obtained in regions of the RET1 gene encoding amino acids 300 to 325, 455 to 486, 487 to 521, and 1061 to 1082 of the protein. In degree of amino acid sequence conservation, these range from highly variable in the first to highly conserved in the last two regions. Residues 300 to 325 yielded mainly reduced-termination mutants, while in region 1061 to 1082, increased-termination mutants were obtained exclusively. All mutants recovered, while causing gain of suppression with one SUP4 allele, brought about a reduction in suppression with the other allele, thus confirming that the phenotype is due to altered termination rather than an elevated level of transcription initiation. In vitro transcription reactions performed with extracts from several strong mutants demonstrated that the mutant polymerases respond to RNA terminator sequences in a manner that matches their in vivo termination phenotypes.

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