DNA-dependent in vitro synthesis of ribosomal proteins, protein elongation factors, and RNA polymerase subunit α: Inhibition by ppGpp

We cloned the genes encoding the ribosomal proteins Ph (Pyrococcus horikoshii)-P0, Ph-L12 and Ph-L11, which constitute the GTPase-associated centre of the archaebacterium Pyrococcus horikoshii. These proteins are homologues of the eukaryotic P0, P1/P2 and eL12 proteins, and correspond to Escherichia coli L10, L7/L12 and L11 proteins respectively. The proteins and the truncation mutants of Ph-P0 were overexpressed in E. coli cells and used for in vitro assembly on to the conserved domain around position 1070 of 23S rRNA (E. coli numbering). Ph-L12 tightly associated as a homodimer and bound to the C-terminal half of Ph-P0. The Ph-P0·Ph-L12 complex and Ph-L11 bound to the 1070 rRNA fragments from the three biological kingdoms in the same manner as the equivalent proteins of eukaryotic and eubacterial ribosomes. The Ph-P0·Ph-L12 complex and Ph-L11 could replace L10·L7/L12 and L11 respectively, on the E. coli 50S subunit in vitro. The resultant hybrid ribosome was accessible for eukaryotic, as well as archaebacterial elongation factors, but not for prokaryotic elongation factors. The GTPase and polyphenylalanine-synthetic activity that is dependent on eukaryotic elongation factors was comparable with that of the hybrid ribosomes carrying the eukaryotic ribosomal proteins. The results suggest that the archaebacterial proteins, including the Ph-L12 homodimer, are functionally accessible to eukaryotic translation factors.

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Oligo(A)-stimulated Tetrahymena rDNA synthesis in vitro

Canadian Journal of Biochemistry ◽

10.1139/o81-055 ◽

1981 ◽

Vol 59 (6) ◽

pp. 396-403 ◽

Cited By ~ 2

Author(s):

Peter R. Ganz ◽

Gyorgy B. Kiss ◽

Ronald E. Pearlman

Keyword(s):

Rna Polymerase ◽

Dna Synthesis ◽

Dna Polymerase ◽

Ribosomal Rna ◽

Replication Proteins ◽

General Applicability ◽

In Vitro Synthesis ◽

Restriction Fragments ◽

Double Stranded Dna

The synthesis of Tetrahymena rDNA has been examined using purified DNA polymerase and partially purified preparations of homologous replication enzymes (fraction IV). DNA synthesis with purified DNA polymerase alone was less than that with fraction IV enzymes. This suggested that there were additional factors in fraction IV other than DNA polymerase which contributed to or enhanced rDNA synthesis in vitro. Neither hybridization of rDNA with Tetrahymena ribosomal RNA nor preincubation of rDNA with homologous or heterologous RNA polymerase served to stimulate in vitro synthesis by fraction IV enzymes. However, when rDNA was hybridized with oligoriboadenylate, DNA synthesis using fraction IV was stimulated approximately 4- to 4.5-fold over 150 min of incubation, relative to a similarly treated but unhybridized rDNA control. Using oligoriboadenylate-hybridized EcoR1 and HindIII restriction fragments of rDNA to localize the synthesis most of the in vitro synthesis occurred within a 2.4 × 106 Mr fragment encompassing the centre of the rDNA molecule. The approach of hybridizing a synthetic homooligoribonucleotide primer to double-stranded DNA should prove to be of general applicability in designing similar template–primers in other systems for the purpose of isolating replication proteins.

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Coupled in vitro synthesis and splicing of RNA polymerase II transcripts

RNA ◽

10.1017/s1355838200992537 ◽

2000 ◽

Vol 6 (9) ◽

pp. 1325-1334 ◽

Cited By ~ 44

Author(s):

SAGARMOY GHOSH ◽

MARIANO A. GARCIA-BLANCO

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

In Vitro Synthesis

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The relationship between the spoT gene, the synthesis of stable RNA, ribosomal proteins, and the ββ′ subunits of RNA polymerase following a nutritional shiftup of Escherichia coli

Canadian Journal of Biochemistry ◽

10.1139/o78-081 ◽

1978 ◽

Vol 56 (6) ◽

pp. 528-533 ◽

Cited By ~ 6

Author(s):

Stephen M. Boyle ◽

Frederick Chu ◽

Nathan Brot ◽

Bruce H. Sells

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Ribosomal Protein ◽

Ribosomal Proteins ◽

Rna Synthesis ◽

Transient Increase ◽

Spot Gene ◽

The Relationship

The level of ppGpp and rates of synthesis of stable RNA, ribosomal protein, and the β and β′ subunits of RNA polymerase were measured following a nutritional shiftup in Escherichia coli strains, NF 929 (spoT+) and NF 930 (spoT'−). In the spoT+ strain, ppGpp levels decreased 50% within 2 min following shiftup, and the rates of synthesis of stable RNA, ribosomal proteins, and the β and β′ subunits of RNA polymerase increased with little or no lag. In contrast, in the spoT− strain, ppGpp levels transiently increased 40% during the first 6 min following shiftup. An inhibition in the rate of stable RNA synthesis and a delay in the increased synthesis of ribosomal proteins and β and β′ subunits occurred concurrently with the transient increase in ppGpp. In addition, the DNA-dependent synthesis in vitro of the β and β′ subunits of RNA polymerase was inhibited by physiological levels of ppGpp. Because of the timing and magnitude of the changes in ppGpp levels in the spoT− strain versus the timing when the new rates of stable RNA, ribosomal protein, and β and β′ subunits synthesis are reached, it is concluded that ppGpp is not the sole element regulating the expression of these genes.

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Arguments based on hybridization-competition experiments in favor of the in vitro synthesis of sporulation-specific mRNAs by the RNA polymerase of B. thuringiensis

Biochemical and Biophysical Research Communications ◽

10.1016/0006-291x(76)90702-6 ◽

1976 ◽

Vol 73 (2) ◽

pp. 263-270 ◽

Cited By ~ 10

Author(s):

AndréF. Klier ◽

Marguerite-M. Lecadet

Keyword(s):

Rna Polymerase ◽

In Vitro Synthesis ◽

Specific Mrnas

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In vitro synthesis of yeast ribosomal proteins

FEBS Letters ◽

10.1016/0014-5793(75)80263-8 ◽

1975 ◽

Vol 58 (1-2) ◽

pp. 219-221 ◽

Cited By ~ 8

Author(s):

W.H. Mager ◽

R. Hoving ◽

R.J. Planta

Keyword(s):

Ribosomal Proteins ◽

In Vitro Synthesis

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In vitro Synthesis of a DNA Dependent RNA Polymerase Coded on Coliphage T7 Genome

Nature ◽

10.1038/2281162a0 ◽

1970 ◽

Vol 228 (5277) ◽

pp. 1162-1165 ◽

Cited By ~ 14

Author(s):

DAVID H. GELFAND ◽

MASAAKI HAYASHI

Keyword(s):

Rna Polymerase ◽

In Vitro Synthesis

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In vitro transcription of viroid RNA into full-length copies by RNA-dependent RNA polymerase from healthy tomato leaf tissue

Bioscience Reports ◽

10.1007/bf01116382 ◽

1982 ◽

Vol 2 (3) ◽

pp. 185-194 ◽

Cited By ~ 17

Author(s):

Frank Boege ◽

Wolfgang Rohde ◽

Heinz L. Sänger

Keyword(s):

Rna Polymerase ◽

Tomato Plant ◽

Leaf Tissue ◽

Potato Spindle Tuber Viroid ◽

In Vitro Transcription ◽

Tomato Leaf ◽

Full Length ◽

Rna Dependent Rna Polymerase ◽

In Vitro Synthesis

RNA-dependent RNA polymerase from healthy tomato plant tissue accepts potato spindle tuber viroid (PSTV) RNA as a template for the in vitro synthesis of full-length RNA copies of the PSTV genome. Viroid transcription requires the presence of Mn2+ and/or Mg2+ ions and is not inhibited by concentrations of 10−5 M α-amanitin. This is the first report of a well-defined product synthesized in vitro by an RNA-dependent RNA polymerase from healthy plants.

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Roles of SigB and SigF in the Mycobacterium tuberculosis Sigma Factor Network

Journal of Bacteriology ◽

10.1128/jb.01273-07 ◽

2007 ◽

Vol 190 (2) ◽

pp. 699-707 ◽

Cited By ~ 57

Author(s):

Jong-Hee Lee ◽

Petros C. Karakousis ◽

William R. Bishai

Keyword(s):

Mycobacterium Tuberculosis ◽

Rna Polymerase ◽

Sigma Factor ◽

Ribosomal Proteins ◽

Consensus Sequence ◽

In Vitro Transcription ◽

Transcription Analysis ◽

Recombinant Strains ◽

Regulatory Cascade

ABSTRACTTo characterize the roles of SigB and SigF in sigma factor regulation inMycobacterium tuberculosis, we used chemically inducible recombinant strains to conditionally overexpresssigBandsigF.Using whole genomic microarray analysis and quantitative reverse transcription-PCR, we investigated the resulting global transcriptional changes aftersigBinduction, and we specifically tested the relative expression of other sigma factor genes after knock-in expression ofsigBandsigF. Overexpression ofsigBresulted in significant upregulation of genes encoding several early culture filtrate antigens (ESAT-6-like proteins), ribosomal proteins, PE-PGRS proteins, the keto-acyl synthase, KasA, and the regulatory proteins WhiB2 and IdeR. Of note, the induction ofsigBdid not alter the expression of other sigma factor genes, indicating that SigB is likely to serve as an end regulator for at least one branch of theM. tuberculosissigma factor regulatory cascade. Analysis of the 5′-untranslated region (UTR) of SigB-dependent transcripts revealed a putative consensus sequence of NGTGG-N14-18-NNGNNG. This sequence appeared upstream of bothsigB(Rv2710) and the gene following it,ideR(Rv2711), and in vitro transcription analysis with recombinant SigB-reconstituted RNA polymerase confirmed SigB-dependent transcription from each of these promoters. Knock-in expression ofsigFrevealed that only thesigCgene was significantly upregulated 6 and 12 h aftersigFinduction. The previously identified SigF promoter consensus sequence AGTTTG-N15-GGGTTT was identified in the 5′ UTR of thesigCgene, and SigF-dependent in vitro transcription of the promoter upstream ofsigCwas confirmed by using recombinant SigF-reconstituted RNA polymerase. These two knock-in recombinant strains were tested in a macrophage model of infection which showed that overexpression ofsigBandsigFresulted in reduced rates ofM. tuberculosisintracellular growth. These results define the SigB promoter consensus recognition sequence and members of the SigB regulon. Moreover, the data suggest that, in addition to serving as an end regulator in a sigma factor cascade, SigB may auto-amplify its own expression under certain conditions.

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