Bacteriophage T4 late promoters: mapping 5′ ends of T4 gene 23 mRNAs.

Kassavetis, George A.; Geiduschek, E. Peter

doi:10.1002/j.1460-2075.1982.tb01132.x

Cited by 69 publications

(25 citation statements)

References 27 publications

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“…Purified oligonucleotide (0.5 to 1.0 ,ug) was 5' end labeled with [y-32P]ATP as described by Maniatis et al (19) with T4 polynucleotide kinase. Total in vivo-synthesized RNA was isolated from a 100-ml midlog culture of C600 grown in TB supplemented with 5 mM oleate-0.5% Brij 58 by a modification of the procedure of Kassavetis and Geiduschek (14). After cell lysis, sample volumes were adjusted to 3.4 ml with 100 mM Tris hydrochloride (pH 8.0)-2 mM EDTA, and 2-mercaptoethanol was added to 1.0%.…”

Section: Methodsmentioning

confidence: 99%

Primary sequence of the Escherichia coli fadL gene encoding an outer membrane protein required for long-chain fatty acid transport

Black

1991

J Bacteriol

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The fadL gene of Escherichia coli encodes an outer membrane protein (FadL) that plays a central role in the uptake of exogenous long-chain fatty acids. The nucleotide sequence of the fadL gene revealed a single open reading frame of 1,344 bp encoding a protein with 448 amino acid residues and a molecular weight of 48,831. The transcriptional start, analyzed by primer extension, was shown to be 95 bp upstream from the translational start. Apparent -10 and -35 regions were found at -12 and -37 bp upstream from the transcriptional start. Three regions with hyphenated dyad symmetry (two between the transcriptional start and the translational start and one upstream from the -10 and -35 regions) were identified that may play a role in the expression offadL. The protein product of the fadL gene contained a signal sequence and signal peptidase I cleavage site similar to that defined for other E. coli outer membrane proteins. The N-terminal sequence of the mature FadL protein was determined by automated amino acid sequencing of protein purified from the outer membrane of a strain harboring fadL under the control of a T7 RNA polymerase-responsive promoter. This amino acid sequence, Ala-Gly-Phe-Gln-Leu-Asn-Glu-Phe-Ser-Ser, verified the signal peptidase I cleavage site on pre-FadL and confirmed the N-terminal amino acid sequence of FadL predicted from the DNA sequence. Mature FadL contained 421 amino acid residues, giving a molecular weight of 45,969. The amino acid composition of FadL deduced from the DNA sequence suggested that this protein contained an abundance of hydrophobic amino acid residues and lacked cysteinyl residues. The hydrophobic amino acids within FadL were predicted to contribute to at least five regions of the protein with an overall hydrophobic character. The amino acid sequence of FadL was used to search GenBank for other proteins with amino acid sequence homology. These data demonstrated that FadL and the heat-modifiable outer membrane protein P1 of Haemophilus influenzae type b were 60.5% conserved and 42.0% identical over 438 amino acid residues.

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Section: Methodsmentioning

confidence: 99%

Primary sequence of the Escherichia coli fadL gene encoding an outer membrane protein required for long-chain fatty acid transport

Black

1991

J Bacteriol

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“…The BamHI site was filled in with Escherichia coli DNA polymerase I Klenow fragment, dGTP, dCTP, dTTP, and [kx-32P]dATP (5,000 Ci/mmol; Dupont, NEN Research Products, Boston, Mass.). The BamHI-PstI fragment containing the tRNA gene insert was excised from a 4% polyacrylamide gel, passively eluted into 10 mM Tris chloride (pH 8.0)-0.2 mM trisodium EDTA-0.1% sodium dodecyl sulfate-i M LiCl, precipitated with ethanol, suspended in 10 mM Tris chloride (pH 8.0)-0.1 mM trisodium EDTA, and purified on benzoylated naphthoylated DEAE-cellulose (35). pTZ1, pTZ2, pSUP6, and pLN4132 were cleaved with EcoRI and PvullI, and the EcoRI site was labeled by filling in with [at-32P]dATP and passed over a 1-ml Sepharose CL-2B column to remove the small labeled EcoRI-Pi,II fragment.…”

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confidence: 99%

Transcription factor IIIB generates extended DNA interactions in RNA polymerase III transcription complexes on tRNA genes.

et al. 1989

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Transcription complexes that assemble on tRNA genes in a crude Saccharomyces cerevisiae cell extract extend over the entire transcription unit and approximately 40 base pairs of contiguous 5'-flanking DNA. We show here that the interaction with 5'-flanking DNA is due to a protein that copurifies with transcription factor TFIIIB through several steps of purification and shares characteristic properties that are normally ascribed to TFIIIB: dependence on prior binding of TFIIIC and great stability once the TFIIIC-TFIIIB-DNA complex is formed. SUP4 gene (tRNATyr) DNA that was cut within the 5'-flanking sequence (either 31 or 28 base pairs upstream of the transcriptional start site) was no longer able to stably incorporate TFIIIB into a transcription complex. The TFIIIB-dependent 5'-flanking DNA protein interaction was predominantly not sequence specific. The extension of the transcription complex into this DNA segment does suggest two possible explanations for highly diverse effects of flanking-sequence substitutions on tRNA gene transcription: either (i) proteins that are capable of binding to these upstream DNA segments are also potentially capable of stimulating or interfering with the incorporation of TFIIIB into transcription complexes or (ii) 5'-flanking sequence influences the rate of assembly of TFIIIB into stable transcription complexes.Specific transcription by RNA polymerase III (Pol III) requires the participation of multiple transcription factors. With the exception of the U6 and 7SK RNA genes (7, 14, 17. 41. 50, 53), the DNA-binding sites that anchor these factors are located within transcription units. It is a remarkable property of Pol III that it can transcribe through the bulky transcription complexes that are built up on these internal promoters (also called internal control regions) without dispersing them (69).Specific initiation of transcription at tRNA genes, with which this paper primarily deals, requires factors TFIIIB and -C. Although the yeast analog of TFIIIC (also called T) has been relatively highly purified as a single component (54: see below), HeLa TFIIIC splits into two components upon purification: C2 is the primary DNA-binding determinant, and Cl either modifies the binding properties of C2 or binds to DNA in a C2-dependent manner (12,18,71). Both HeLa C2 factor and yeast TFIIIC (T) are large (12,54,64). The Bonbvx moni tRNA gene transcription factors can also be separated into three fractions, whose relationship to TFIIIB, Cl, and C2 remains to be established (51).The first promoter dissections of Pol III genes identified essential gene-internal elements (10,19,25,55) and implied that flanking DNA sequence was almost without effect on promoter strength. The general situation with regard to the effects of flanking sequence on promoter strength of Pol III genes is, however, diverse, and this fact has only gradually been recognized (3, 4, 20, 29, 52, 62, 63; tions with very substantial effects have been reported. Flanking sequences affecting promoter strength are located within ...

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“…S1 nuclease mapping was performed as described by Burke (3). In the primer extension experiments (22), we used three different oligonucleotide primers that corresponded to positions 446 to 465 (primer 316:…”

Section: Methodsmentioning

confidence: 99%

Discovery of a rhizobial RNA that is essential for symbiotic root nodule development

1991

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All of the Azorhizobium, Bradyrhizobium, and Rhizobium genes known to be involved in the development of nitrogen-fixing legume root nodules are genes that code for proteins. Here we report the first exception to this rule: the sra gene; it was discovered during the genetic analysis of a Bradyrhizobium japonicum TnS mutant (strain 259) which had a severe deficiency in colonizing soybean nodules. A DNA region as small as 0.56 kb cloned from the parental wild type restored a wild-type phenotype in strain 259 by genetic complementation. The sra gene was located on this fragment, sequenced, and shown to be transcribed into a 213-nucleotide RNA. Results obtained with critical point mutations in the sra gene proved that the transcript was not translated into protein; rather, it appeared to function as an RNA molecule with a certain stem-and-loop secondary structure. We also detected an sra homolog in Rhizobium meliloti which, when cloned and transferred to B. japonicum mutant 259, fully restored symbiotic effectiveness in that strain. We propose several alternative functions for the sra gene product, of which that as a regulatory RNA for gene expression may be the most probable one.

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Bacteriophage T4 late promoters: mapping 5′ ends of T4 gene 23 mRNAs.

Cited by 69 publications

References 27 publications

Primary sequence of the Escherichia coli fadL gene encoding an outer membrane protein required for long-chain fatty acid transport

Primary sequence of the Escherichia coli fadL gene encoding an outer membrane protein required for long-chain fatty acid transport

Transcription factor IIIB generates extended DNA interactions in RNA polymerase III transcription complexes on tRNA genes.

Discovery of a rhizobial RNA that is essential for symbiotic root nodule development

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