An in vitro transcription termination system to analyze chloroplast promoters: identification of multiple promoters for the spinach atpB gene

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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The NtcA-Dependent P1 Promoter Is Utilized for glnA Expression in N2-Fixing Heterocysts of Anabaena sp. Strain PCC 7120

Journal of Bacteriology ◽

10.1128/jb.186.21.7337-7343.2004 ◽

2004 ◽

Vol 186 (21) ◽

pp. 7337-7343 ◽

Cited By ~ 36

Author(s):

Ana Valladares ◽

Alicia M. Muro-Pastor ◽

Antonia Herrero ◽

Enrique Flores

Keyword(s):

Binding Site ◽

In Vitro Transcription ◽

Upstream Region ◽

Gene Encoding ◽

Differentiated Cells ◽

Multiple Promoters ◽

Key Enzyme ◽

Anabaena Sp ◽

Pcc 7120

ABSTRACT Expression of the glnA gene encoding glutamine synthetase, a key enzyme in nitrogen metabolism, is subject to a variety of regulatory mechanisms in different organisms. In the filamentous, N2-fixing cyanobacterium Anabaena sp. strain PCC 7120, glnA is expressed from multiple promoters that generate several transcripts whose abundance is influenced by NtcA, the transcription factor exerting global nitrogen control in cyanobacteria. Whereas RNAI originates from a canonical NtcA-dependent promoter (P1) and RNAII originates from a σ70-type promoter (P2), RNAIV is influenced by NtcA but the corresponding promoter (P3) does not have the structure of NtcA-activated promoters. Using RNA isolated from Anabaena filaments grown under different nitrogen regimens, we observed, in addition to these transcripts, RNAV, which has previously been detected only in in vitro transcription assays and should originate from P4. However, in heterocysts, which are differentiated cells specialized in N2 fixation, RNAI was the almost exclusive glnA transcript. Analysis of P glnA ::lacZ fusions containing different fragments of the glnA upstream region confirmed that fragments carrying P1, P2, or P3 and P4 have the ability to promote transcription. Mutation of the NtcA-binding site in P1 eliminated P1-directed transcription and allowed increased use of P2. The NtcA-binding site in the P1 promoter and binding of NtcA to this site appear to be key factors in determining glnA gene expression in vegetative cells and heterocysts.

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A tridecamer DNA sequence supports human mitochondrial RNA 3'-end formation in vitro.

Molecular and Cellular Biology ◽

10.1128/mcb.8.10.4502 ◽

1988 ◽

Vol 8 (10) ◽

pp. 4502-4509 ◽

Cited By ~ 59

Author(s):

T W Christianson ◽

D A Clayton

Keyword(s):

Dna Sequence ◽

Transcription Termination ◽

In Vitro Transcription ◽

Mitochondrial Rna ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Transcription System ◽

Flanking Regions

Vertebrate mitochondrial genomes contain a putative transcription termination site at the boundary between the genes for 16S rRNA and leucyl-tRNA. We have described previously an in vitro transcription system from human cells with the capacity to generate RNA 3' ends with the same map positions as those synthesized in vivo. By assaying the ability of variously truncated templates to support 3'-end formation, we demonstrated that the tridecamer sequence 5'-TGGCAGAGCCCCGG-3', contained entirely within the gene for leucyl-tRNA, is necessary to direct accurate termination. When two tridecamer sequences and their immediate flanking regions were placed in tandem, termination occurred at both promoter-proximal and promoter-distal sites. Furthermore, termination was competitively inhibited, in a concentration-dependent manner, by DNA containing the tridecamer sequence. These results suggest a modest sequence requirement for transcription termination that is contingent on a factor capable of recognizing the presence of the tridecamer DNA sequence.

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Regulatory region of the Klebsiella aerogenes tryptophan operon

Journal of Bacteriology ◽

10.1128/jb.152.1.49-56.1982 ◽

1982 ◽

Vol 152 (1) ◽

pp. 49-56

Author(s):

M Blumenberg ◽

C Yanofsky

Keyword(s):

Transcription Termination ◽

Regulatory Region ◽

Enteric Bacteria ◽

In Vitro Transcription ◽

Regulatory Sequences ◽

Klebsiella Aerogenes ◽

Transcription Termination Site ◽

Tryptophan Operon ◽

Trp Promoter

The trp operon of Klebsiella aerogenes was cloned, and its regulatory region was sequenced. Comparison with previously reported trp regulatory sequences of other enteric bacteria indicates that the K. aerogenes trp promoter-operator region is most similar to the corresponding region of Salmonella typhimurium. The trp leader regions of K. aerogenes and other enteric bacteria are organized similarly, but there are significant differences in the stabilities of the predicted secondary structures in their leader transcripts. These differences should make the K. aerogenes attenuator a weaker transcription termination site than any of the other attenuator regions studied; this was confirmed in in vitro transcription experiments. The sequence of the leader transcript and the precise site of in vitro termination were determined.

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A tridecamer DNA sequence supports human mitochondrial RNA 3'-end formation in vitro

Molecular and Cellular Biology ◽

10.1128/mcb.8.10.4502-4509.1988 ◽

1988 ◽

Vol 8 (10) ◽

pp. 4502-4509

Author(s):

T W Christianson ◽

D A Clayton

Keyword(s):

Dna Sequence ◽

Transcription Termination ◽

In Vitro Transcription ◽

Mitochondrial Rna ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Transcription System ◽

Flanking Regions

Vertebrate mitochondrial genomes contain a putative transcription termination site at the boundary between the genes for 16S rRNA and leucyl-tRNA. We have described previously an in vitro transcription system from human cells with the capacity to generate RNA 3' ends with the same map positions as those synthesized in vivo. By assaying the ability of variously truncated templates to support 3'-end formation, we demonstrated that the tridecamer sequence 5'-TGGCAGAGCCCCGG-3', contained entirely within the gene for leucyl-tRNA, is necessary to direct accurate termination. When two tridecamer sequences and their immediate flanking regions were placed in tandem, termination occurred at both promoter-proximal and promoter-distal sites. Furthermore, termination was competitively inhibited, in a concentration-dependent manner, by DNA containing the tridecamer sequence. These results suggest a modest sequence requirement for transcription termination that is contingent on a factor capable of recognizing the presence of the tridecamer DNA sequence.

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Evolutionary changes of sequences and factors that direct transcription termination of human and mouse ribsomal genes.

Molecular and Cellular Biology ◽

10.1128/mcb.7.7.2521 ◽

1987 ◽

Vol 7 (7) ◽

pp. 2521-2529 ◽

Cited By ~ 18

Author(s):

I Bartsch ◽

C Schoneberg ◽

I Grummt

Keyword(s):

Base Pair ◽

Signal Sequence ◽

Transcription Termination ◽

Proximal Part ◽

In Vitro Transcription ◽

Transcription Unit ◽

Rdna Transcription ◽

Human Sequence ◽

Sequence Elements

We have analyzed the sequences required for termination of human rDNA transcription. The human ribosomal transcription unit is shown to extend about 350 nucleotides into the 3'-terminal spacer and ends immediately upstream of a region with a distinct sequence heterogeneity. This heterogeneous region contains a cluster of conserved 10-base pair sequence elements which exert a striking homology to the proximal part of the 18-base pair murine rDNA transcription termination signal sequence, termed SalI box. Exonuclease III protection assays and in vitro transcription experiments with both homologous and heterologous human-mouse minigene constructs, and extracts from HeLa or Ehrlich ascites cells, reveal a functional analogy of the human sequence to the mouse SalI box. It mediates binding of a nuclear protein which functions as a transcription termination factor. The murine signal sequence is recognized by the human factor but not vice versa. The different sequence specificities and electrophoretic properties of the functionally equivalent protein factors suggest that a molecular coevolution has taken place between the termination signal sequences and the genes coding for the termination factors.

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An integrated model for termination of RNA polymerase III transcription

10.1101/2021.08.27.457903 ◽

2021 ◽

Author(s):

Juanjuan Xie ◽

Umberto Aiello ◽

Yves Clement ◽

Nouhou Haidara ◽

Mathias Girbig ◽

...

Keyword(s):

Rna Polymerase ◽

Transcription Termination ◽

Rna Polymerase Iii ◽

In Vitro Transcription ◽

Cis Acting ◽

Template Strand ◽

Polymerase Iii ◽

Genome Wide ◽

Fail Safe

RNA polymerase III (RNAPIII) synthesizes essential and abundant non-coding RNAs such as tRNAs. Controlling RNAPIII span of activity by accurate and efficient termination is a challenging necessity to ensure robust gene expression and to prevent conflicts with other DNA-associated machineries. The mechanism of RNAPIII termination is believed to be simpler than that of other eukaryotic RNA polymerases, solely relying on the recognition of a T-tract in the non-template strand. Here we combine high-resolution genome-wide analyses and in vitro transcription termination assays to revisit the mechanism of RNAPIII transcription termination in budding yeast. We show that T-tracts are necessary but not always sufficient for termination and that secondary structures of the nascent RNAs are important auxiliary cis-acting elements. Moreover, we show that the helicase Sen1 plays a key role in a fail-safe termination pathway. Our results provide a comprehensive model illustrating how multiple mechanisms cooperate to ensure efficient RNAPIII transcription termination.

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Early In Vitro Transcription Termination in Human H5 Influenza Viral RNA Synthesis

Applied Biochemistry and Biotechnology ◽

10.1007/s12010-010-9152-4 ◽

2011 ◽

Vol 164 (4) ◽

pp. 497-513 ◽

Cited By ~ 1

Author(s):

Matthew B. Kerby ◽

Aartik A. Sarma ◽

Madhukar S. Patel ◽

Andrew W. Artenstein ◽

Steven M. Opal ◽

...

Keyword(s):

Rna Synthesis ◽

Transcription Termination ◽

In Vitro Transcription ◽

Viral Rna

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Sequences required for transcription termination at the intrinsic λtI terminator

Canadian Journal of Microbiology ◽

10.1139/w09-123 ◽

2010 ◽

Vol 56 (2) ◽

pp. 168-177 ◽

Cited By ~ 2

Author(s):

Miguel Martínez-Trujillo ◽

Alejandra Sánchez-Trujillo ◽

Víctor Ceja ◽

Federico Ávila-Moreno ◽

Rosa María Bermúdez-Cruz ◽

...

Keyword(s):

Northern Blot ◽

Transcription Termination ◽

In Vitro Transcription ◽

Deletion Mutants ◽

Typical Structure ◽

S1 Mapping ◽

The Stability ◽

Stem Structure

The λtI terminator is located approximately 280 bp beyond the λint gene, and it has a typical structure of an intrinsic terminator. To identify sequences required for λtI transcription termination a set of deletion mutants were generated, either from the 5′ or the 3′ end onto the λtI region. The termination efficiency was determined by measuring galactokinase (galK) levels by Northern blot assays and by in vitro transcription termination. The importance of the uridines and the stability of the stem structure in the termination were demonstrated. The nontranscribed DNA beyond the 3′ end also affects termination. Additionally, sequences upstream have a small effect on transcription termination. The in vivo RNA termination sites at λtI were determined by S1 mapping and were located at 8 different positions. Processing of transcripts from the 3′ end confirmed the importance of the hairpin stem in protection against exonuclease.

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