CCA initiation boxes without unique promoter elements support in vitro transcription by three viral RNA-dependent RNA polymerases

Efficient transcription from the rat rDNA promoter results from an undefined interaction between the core (CPE) and upstream (UPE) promoter elements or the protein complexes which form on them. These interactions were demonstrated by the behavior of promoters that contained either linker-scanning or deletion mutations of the UPE in combination with point mutations of the CPE (bidomain mutants). In vivo transcription experiments using point mutations within the CPE (G----A mutation at either -16 or -7) demonstrated that the CPE may in fact consist of two domains. Whereas both of these mutants were rescued by the addition of UBF to in vitro transcription reactions, the CPE mutant -7A/G was inactive in vivo. Experiments with these bidomain mutants demonstrated that the UPE was required for the rescue of the CPE mutants. We also examined the hypothesis that this interaction might require a stereospecific alignment of the promoter elements. Our results indicate that the promoter consists of several domains with differing responses to mutations that alter the distance between, or within, the promoter elements. For example, the insertion or deletion of half-multiples of the helical repeat distance between -167 and -147 had no significant effect on transcription. On the other hand, some sites were sensitive to deletions of any size but not to insertions of up to 20 bp. The analyses of two sites yielded results suggesting that they lay between domains of the promoter that must be on the same side of the DNA helix for promoter activity. The first of these sites mapped between -106 and -95.(ABSTRACT TRUNCATED AT 250 WORDS)

Download Full-text

cDNA cloning and in vitro transcription of the complete brome mosaic virus genome

Molecular and Cellular Biology ◽

10.1128/mcb.4.12.2876-2882.1984 ◽

1984 ◽

Vol 4 (12) ◽

pp. 2876-2882 ◽

Cited By ~ 1

Author(s):

P Ahlquist ◽

M Janda

Keyword(s):

Mosaic Virus ◽

Transcription Initiation ◽

Direct Sequencing ◽

In Vitro Transcription ◽

Brome Mosaic Virus ◽

Viral Rna ◽

In Vitro Translation ◽

In Vitro Production ◽

Genomic Rnas

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.

Download Full-text

Mutational studies of archaeal RNA polymerase and analysis of hybrid RNA polymerases

Biochemical Society Transactions ◽

10.1042/bst0370018 ◽

2009 ◽

Vol 37 (1) ◽

pp. 18-22 ◽

Cited By ~ 9

Author(s):

Michael Thomm ◽

Christoph Reich ◽

Sebastian Grünberg ◽

Souad Naji

Keyword(s):

Complex Formation ◽

Structural Similarity ◽

In Vitro Transcription ◽

Rna Polymerases ◽

Hyperthermophilic Archaea ◽

Active Centre ◽

Α Subunit ◽

Recent Success ◽

Open Complex Formation

The recent success in reconstitution of RNAPs (RNA polymerases) from hyperthermophilic archaea from bacterially expressed purified subunits opens the way for detailed structure–function analyses of multisubunit RNAPs. The archaeal enzyme shows close structural similarity to eukaryotic RNAP, particularly to polymerase II, and can therefore be used as model for analyses of the eukaryotic transcriptional machinery. The cleft loops in the active centre of RNAP were deleted and modified to unravel their function in interaction with nucleic acids during transcription. The rudder, lid and fork 2 cleft loops were required for promoter-directed initiation and elongation, the rudder was essential for open complex formation. Analyses of transcripts from heteroduplex templates containing stable open complexes revealed that bubble reclosure is required for RNA displacement during elongation. Archaeal transcription systems contain, besides the orthologues of the eukaryotic transcription factors TBP (TATA-box-binding protein) and TF (transcription factor) IIB, an orthologue of the N-terminal part of the α subunit of eukaryotic TFIIE, called TFE, whose function is poorly understood. Recent analyses revealed that TFE is involved in open complex formation and, in striking contrast with eukaryotic TFIIE, is also present in elongation complexes. Recombinant archaeal RNAPs lacking specific subunits were used to investigate the functions of smaller subunits. These studies revealed that the subunits P and H, the orthologues of eukaryotic Rpb12 and Rpb5, were not required for RNAP assembly. Subunit P was essential for open complex formation, and the ΔH enzyme was greatly impaired in all assays, with the exception of promoter recruitment. Recent reconstitution studies indicate that Rpb12 and Rpb5 can be incorporated into archaeal RNAP and can complement for the function of the corresponding archaeal subunit in in vitro transcription assays.

Download Full-text

Inexpensive and colorimetric RNA detection by E. coli cell-free protein synthesis platform at room temperature

10.1101/2021.11.29.21267025 ◽

2021 ◽

Author(s):

Michela Notarangelo ◽

Alessandro Quattrone ◽

Massimo Pizzato ◽

Sheref S. Mansy ◽

O. Duhan Toparlak

Keyword(s):

Room Temperature ◽

Colorimetric Detection ◽

In Vitro Transcription ◽

Viral Rna ◽

Rna Sequences ◽

Rna Detection ◽

E Coli ◽

Free Gene ◽

Cell Free Protein Synthesis

We report colorimetric detection of SARS-CoV-2 viral RNA by an in vitro transcription/translation assay with crude E. coli extracts at room temperature, with the aid of body heat. Clinically-relevant concentrations of viral RNA (ca. 600 copies/test) were detected from synthetic RNA samples. The activation of cell-free gene expression was achieved by toehold-switch-mediated riboregulatory elements that are specific to viral RNA sequences. The colorimetric output was generated by the α-complementation of β-galactosidase ω-fragment (LacZ-ω) with cell-free expressed LacZ-α, using an X-gal analogue as a substrate. The estimated cost of single reaction is less than 1 euro/test, which may facilitate diagnostic kit accessibility in developing countries.

Download Full-text

Hybrid Bordetella pertussis-Escherichia coli RNA Polymerases: Selectivity of Promoter Activation

Journal of Bacteriology ◽

10.1128/jb.180.6.1567-1569.1998 ◽

1998 ◽

Vol 180 (6) ◽

pp. 1567-1569 ◽

Cited By ~ 9

Author(s):

Pierre Steffen ◽

Agnes Ullmann

Keyword(s):

Escherichia Coli ◽

Bordetella Pertussis ◽

In Vitro Transcription ◽

Rna Polymerases ◽

Α Subunit ◽

E Coli ◽

Transcription System ◽

Catabolite Gene Activator Protein ◽

Transcription Activators

ABSTRACT We constructed hybrid Bordetella pertussis-Escherichia coli RNA polymerases and compared productive interactions between transcription activators and cognate RNA polymerase subunits in an in vitro transcription system. Virulence-associated genes of B. pertussis, in the presence of their activator BvgA, are transcribed by all variants of hybrid RNA polymerases, whereas transcription at the E. coli lacpromoter regulated by the cyclic AMP-catabolite gene activator protein has an absolute requirement for the E. coli α subunit. This suggests that activator contact sites involve a high degree of selectivity.

Download Full-text

Mutational Analysis of the Chlamydia trachomatis rRNA P1 Promoter Defines Four Regions Important for Transcription In Vitro

Journal of Bacteriology ◽

10.1128/jb.180.9.2359-2366.1998 ◽

1998 ◽

Vol 180 (9) ◽

pp. 2359-2366 ◽

Cited By ~ 26

Author(s):

Ming Tan ◽

Tamas Gaal ◽

Richard L. Gourse ◽

Joanne N. Engel

Keyword(s):

Escherichia Coli ◽

Chlamydia Trachomatis ◽

Rna Polymerase ◽

Mutational Analysis ◽

In Vitro Transcription ◽

Rna Polymerases ◽

Rich Region ◽

E Coli ◽

Transcription In Vitro

ABSTRACT We have characterized the Chlamydia trachomatisribosomal promoter, rRNA P1, by measuring the effect of substitutions and deletions on in vitro transcription with partially purifiedC. trachomatis RNA polymerase. Our analyses indicate that rRNA P1 contains potential −10 and −35 elements, analogous toEscherichia coli promoters recognized by E-ς70. We identified a novel AT-rich region immediately downstream of the −35 region. The effect of this region was specific for C. trachomatis RNA polymerase and strongly attenuated by single G or C substitutions. Upstream of the −35 region was an AT-rich sequence that enhanced transcription by C. trachomatis and E. coli RNA polymerases. We propose that this region functions as an UP element.

Download Full-text