Electron microscopic mapping and sequence analysis of the terminator for the early message of E. coli phage T7

Priess, Harro; Koller, Barbara; Hess, Barbara; Delius, Hajo

doi:10.1007/bf00267209

Cited by 19 publications

(12 citation statements)

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“…Our data are in good agreement with these sequences (3-7~ higher for the triplet of segments 7, 8 and 9; 3"9~o lower for segment 11 of bovine rotavirus assuming its real number of bp to be very close to that of segment 11 of the human Wa strain). Priess et al (1980) found a linear relationship between length and number of bp of dsDNA in the range of 400 to 10000 bp and there is no reason that this should not be the case for the size range of dsRNAs investigated (Vasquez & Kleinschmidt, 1968). Our measurements agree, with a difference of ~< 6~ (Table 1, column 8), with the cDNA size estimates of size classes II (segments 5 and 6), III (segments 7 to 9) and IV (segments 10 and 11) (Both et al, 1982), whereas they are 8 to 17 ~ lower for molecules of size class I (segments 1 to 4).…”

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“…The sequence of the $2 gene of reovirus (Cashdollar et al, 1982) confirms the corresponding part of these data. From measurements of sequenced dsDNA standards by electron microscopy (Priess et al, 1980) a number of 1.91 × 106 mol. wt.…”

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confidence: 99%

“…of dsDNA per unit length (~tm) was calculated, which is 10~ below the assumed value for dsRNA (Bellamy et al, 1967). Priess et al (1980) found that the lengths of DN AmRNA hybrids of Escherichia coli phage T7 are shorter than the corresponding dsDNAs and had to be multiplied by a factor of 1.15 in order to calculate the correct number of bps present. These differences obtained from measurements by electron microscopy are in the same size range as those obtained from physicochemical studies (Cantor & Schimmel, 1980) which we have taken as a basis for calculations comparing lengths of dsDNAs and dsRNAs measured in one preparation.…”

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Rotavirus RNA Segments Sized by Electron Microscopy

Rixon

Taylor

Desselberger

1984

Journal of General Virology

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Rotavirus RNA Segments Sized by Electron Microscopy

Rixon

Taylor

Desselberger

1984

Journal of General Virology

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“…Purified inserts were then denatured and hybridized to 428 or 26S RNA in a buffer containing 80% (vol/vol) formamide (29). The nucleic acids were treated with gene 32 protein of the T4 phage and adsorbed to mica sheets (30). The length of the insert molecule and its location (that is, its distance from the 3' end of the RNA molecule) were measured.…”

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The capsid protein of Semliki Forest virus has clusters of basic amino acids and prolines in its amino-terminal region.

Garoff¹,

Frischauf²,

Simons³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

126

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The amino acid sequence of the capsid (C) protein was deduced from the nucleotide sequence of the C gene. This part of the viral 42S RNA genome was transcribed into double-stranded cDNA. The cDNA was cloned in the Escherichia coil X1776-pBR322 host-vector system and then the base sequence was determined with the technique described by Maxam and Gilbert. The amino acid sequence of the C protein shows a clustering of basic amino acids and prolines within the first 110 amino acids.from the virus. This specificity can probably be explained by the formation of bonds between the C protein in the nucleocapsid and the spanning segments of the spike glycoproteins (9).A more detailed understanding of SFV structure and assembly at the molecular level is difficult without the knowledge of the amino acid sequences of the structural proteins. We report here the primary structure of the C protein.Semliki Forest virus (SFV) is a simple membrane virus of the alphavirus group. It has been used extensively as a model system to study the structure and assembly of cellular membranes (1). The virus particle consists of an icosahedral nucleocapsid surrounded by a membrane. The nucleocapsid is a complex of about 240 capsid proteins (C protein, Mr = 30,000) (2, 3) and a RNA molecule (42S), the viral genome (4). The membrane consists of a lipid bilayer with about 240 external glycoprotein spikes (5). Each spike contains three different glycopolypeptides: El (Mr = 49,000), E2 (Mr = 52,000), and E3 (Mr = 10,000) (6, 7). The E2 polypeptide spans the membrane; there are about 30 amino acid residues present on the internal side of the viral membrane (8, 9).The virus enters the host cell by absorptive endocytosis (10). Inside the lysosomes of the cell, the low pH probably triggers a fusion of the viral membrane with the lysosomal membrane (10,11). This allows the nucleocapsid to enter the cell cytoplasm, where the viral genome is uncoated so that it can act as a mRNA for synthesis of polymerase molecules. The viral RNA polymerase synthesizes new 42S RNA molecules and smaller 26S RNA molecules. The latter molecule is homologous to the 3' end of the viral genome (12) and functions as a mRNA for the SFV structural proteins, which are translated from a single initiation site (13). The C protein is made first. As soon as it is completed it is cleaved from the growing polypeptide chain, and the ribosomes continue to read off the membrane proteins in the order E3, E2, and El (8, 14). The membrane proteins are cotranslationally translocated across the membrane of the endoplasmic reticulum and transported to the plasma membrane (15, 16).The assembly of the nucleocapsid in the cell cytoplasm is not understood. Newly synthesized capsid proteins are known to be associated with the large subunit of the ribosome before they complex with the 42S RNA into nucleocapsids (17). The final step in SFV assembly, budding, takes place at the cell surface (18). The nucleocapsid binds to the cytoplasmic aspect of the plasma membrane which folds around the nu...

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“…The tl terminator has recently been accurately mapped, by DNA and RNA sequence studies, at 18.97% from the left end of the DNA (2,15,16).…”

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Termination of transcription of the coliphage T7 “early” operon in vitro: slowness of enzyme release, and lack of any role for sigma

O’Hare

Hayward

1981

Nucl Acids Res

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The leftmost portion of the coliphage T7 genome is transcribed by the RNA polymerase Escherichia coli immediately after infection.This "early" operon is delineated by three promoters on the left, and a transcriptional terminator on the right.The terminator is efficient both in vivo, and with highly purified RNA polymerase in vitro.We have studied termination in vitro, using synchronously initiated transcription reactions with low enzyme:DNA ratios.We show that recognition of the stop signal and release of RNA product are relatively rapid.Dissociation of the enzyme from the DNA is quite slow, and probably rate-limiting for re-cycling of the polymeras.It is well established that the sigma subunit of RNA polymerase is required for specific initiation, but redundant during RNA elongation. By exploiting antisigma antiserum we have abtained evidence that sigma is also redundant during all steps of termination in vitro.

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Electron microscopic mapping and sequence analysis of the terminator for the early message of E. coli phage T7

Cited by 19 publications

References 31 publications

Rotavirus RNA Segments Sized by Electron Microscopy

Rotavirus RNA Segments Sized by Electron Microscopy

The capsid protein of Semliki Forest virus has clusters of basic amino acids and prolines in its amino-terminal region.

Termination of transcription of the coliphage T7 “early” operon in vitro: slowness of enzyme release, and lack of any role for sigma

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