scholarly journals Silkworm 5S RNA and Alanine tRNA Genes Share Highly Conserved 5′ Flanking and Coding Sequences

1982 ◽  
Vol 2 (12) ◽  
pp. 1524-1531 ◽  
Author(s):  
Diane G. Morton ◽  
Karen U. Sprague
Keyword(s):  
Transcription Initiation ◽  
Genomic Dna ◽  
Structural Features ◽  
In Vitro Transcription ◽  
Trna Genes ◽  
5S Rna ◽  
Coding Sequences ◽  
Coding Regions ◽  
The One

A fragment ofBombyx morigenomic DNA containing one tRNA2Alagene and one 5S RNA gene has been used to compare the structural features of silkworm 5S RNA and tRNA genes. The nucleotide sequences of both genes and of the primary transcripts produced from them in homologous in vitro transcription systems have been determined. Comparison of the sequences of these two genes with that of another previously analyzedB. moritRNA2Alagene reveals common oligonucleotides which may be important transcriptional signals. The oligonucleotides TA(C)TAT, AATTTT, and TTC are located approximately (±1 nucleotide) 29, 19, and 3 nucleotides, respectively, before the transcription initiation sites of the two tRNA2Alagenes and the one 5S RNA gene we have analyzed. The sequence GGGCGTAG(C)TCAG lies within the coding regions of all three genes. The functional significance of these sequences is suggested by their location within regions required for the transcription of silkworm alanine tRNA genes in vitro.

Silkworm 5S RNA and Alanine tRNA Genes Share Highly Conserved 5′ Flanking and Coding Sequences

1982 ◽  
Vol 2 (12) ◽  
pp. 1524-1531
Author(s):  
Diane G. Morton ◽  
Karen U. Sprague
Keyword(s):  
Transcription Initiation ◽  
Genomic Dna ◽  
Structural Features ◽  
In Vitro Transcription ◽  
Trna Genes ◽  
5S Rna ◽  
Coding Sequences ◽  
Coding Regions ◽  
The One

A fragment of Bombyx mori genomic DNA containing one tRNA 2 Ala gene and one 5S RNA gene has been used to compare the structural features of silkworm 5S RNA and tRNA genes. The nucleotide sequences of both genes and of the primary transcripts produced from them in homologous in vitro transcription systems have been determined. Comparison of the sequences of these two genes with that of another previously analyzed B. mori tRNA 2 Ala gene reveals common oligonucleotides which may be important transcriptional signals. The oligonucleotides TA(C)TAT, AATTTT, and TTC are located approximately (±1 nucleotide) 29, 19, and 3 nucleotides, respectively, before the transcription initiation sites of the two tRNA 2 Ala genes and the one 5S RNA gene we have analyzed. The sequence GGGCGTAG(C)TCAG lies within the coding regions of all three genes. The functional significance of these sequences is suggested by their location within regions required for the transcription of silkworm alanine tRNA genes in vitro.

scholarly journals Formation of an active transcription complex in the Drosophila melanogaster 5S RNA gene is dependent on an upstream region.

1987 ◽  
Vol 7 (6) ◽  
pp. 2046-2051 ◽  
Author(s):  
A D Garcia ◽  
A M O'Connell ◽  
S J Sharp
Keyword(s):  
Drosophila Melanogaster ◽  
Transcription Initiation ◽  
Rna Polymerase Iii ◽  
In Vitro Transcription ◽  
Upstream Region ◽  
Nucleotide Position ◽  
Wild Type ◽  
5S Rna ◽  
Cell Extracts

We constructed deletion-substitution and linker-scanning mutations in the 5'-flanking region of the Drosophila melanogaster 5S RNA gene. In vitro transcription of these templates in Drosophila and HeLa cell extracts revealed the presence of an essential control region (-30 region) located between nucleotides -39 and -26 upstream of the transcription initiation site: deletion of sequences upstream of nucleotide position -39 had no detectable effect on the wild-type level of in vitro transcription, whereas mutations extending between positions -39 and 1 resulted in templates with decreased transcriptional levels; specifically, deletion and linker-scanning mutations in the -34 to -26 region (-30 region) resulted in loss of transcription. The -30 region is essential for transcription and therefore forms part of the Drosophila 5S RNA gene transcription promoter. Compared with the activity of the wild-type gene, mutant 5S DNAs exhibited no impairment in the ability to sequester limiting transcription factors in a template exclusion competition assay. While we do not know which transcription factor(s) interacts with the -30 region, the possible involvement of RNA polymerase III at this region is discussed.

Formation of an active transcription complex in the Drosophila melanogaster 5S RNA gene is dependent on an upstream region

1987 ◽  
Vol 7 (6) ◽  
pp. 2046-2051
Author(s):  
A D Garcia ◽  
A M O'Connell ◽  
S J Sharp
Keyword(s):  
Drosophila Melanogaster ◽  
Transcription Initiation ◽  
Rna Polymerase Iii ◽  
In Vitro Transcription ◽  
Upstream Region ◽  
Nucleotide Position ◽  
Wild Type ◽  
5S Rna ◽  
Cell Extracts

We constructed deletion-substitution and linker-scanning mutations in the 5'-flanking region of the Drosophila melanogaster 5S RNA gene. In vitro transcription of these templates in Drosophila and HeLa cell extracts revealed the presence of an essential control region (-30 region) located between nucleotides -39 and -26 upstream of the transcription initiation site: deletion of sequences upstream of nucleotide position -39 had no detectable effect on the wild-type level of in vitro transcription, whereas mutations extending between positions -39 and 1 resulted in templates with decreased transcriptional levels; specifically, deletion and linker-scanning mutations in the -34 to -26 region (-30 region) resulted in loss of transcription. The -30 region is essential for transcription and therefore forms part of the Drosophila 5S RNA gene transcription promoter. Compared with the activity of the wild-type gene, mutant 5S DNAs exhibited no impairment in the ability to sequester limiting transcription factors in a template exclusion competition assay. While we do not know which transcription factor(s) interacts with the -30 region, the possible involvement of RNA polymerase III at this region is discussed.

A discrete region centered 22 base pairs upstream of the initiation site modulates transcription of Drosophila tRNAAsn genes

1988 ◽  
Vol 8 (10) ◽  
pp. 4441-4449
Author(s):  
A K Lofquist ◽  
A D Garcia ◽  
S J Sharp
Keyword(s):  
Transcription Initiation ◽  
In Vitro Transcription ◽  
Initiation Site ◽  
Trna Genes ◽  
Selection Mechanism ◽  
Base Pairs ◽  
Discrete Region ◽  
Flanking Sequences ◽  
Actual Sequence

We have studied the mechanism by which 5'-flanking sequences modulate the in vitro transcription of eucaryotic tRNA genes. Using deletion and linker substitution mutagenesis, we have found that the 5'-flanking sequences responsible for the different in vitro transcription levels of three Drosophila tRNA5Asn genes are contained within a discrete region centered 22 nucleotides upstream from the transcription initiation site. In conjunction with the A-box intragenic control region, this upstream transcription-modulatory region functions in the selection mechanism for the site of transcription initiation. Since the transcription-modulatory region directs the position of the start site and the actual sequence of the transcription-modulatory region determines the level of tRNAAsn gene transcription, the possibility is raised that the transcription-modulatory region directs a transcription initiation event similar to open complex formation at procaryotic promoters.

scholarly journals A discrete region centered 22 base pairs upstream of the initiation site modulates transcription of Drosophila tRNAAsn genes.

1988 ◽  
Vol 8 (10) ◽  
pp. 4441-4449 ◽  
Author(s):  
A K Lofquist ◽  
A D Garcia ◽  
S J Sharp
Keyword(s):  
Transcription Initiation ◽  
In Vitro Transcription ◽  
Initiation Site ◽  
Trna Genes ◽  
Selection Mechanism ◽  
Base Pairs ◽  
Discrete Region ◽  
Flanking Sequences ◽  
Actual Sequence

We have studied the mechanism by which 5'-flanking sequences modulate the in vitro transcription of eucaryotic tRNA genes. Using deletion and linker substitution mutagenesis, we have found that the 5'-flanking sequences responsible for the different in vitro transcription levels of three Drosophila tRNA5Asn genes are contained within a discrete region centered 22 nucleotides upstream from the transcription initiation site. In conjunction with the A-box intragenic control region, this upstream transcription-modulatory region functions in the selection mechanism for the site of transcription initiation. Since the transcription-modulatory region directs the position of the start site and the actual sequence of the transcription-modulatory region determines the level of tRNAAsn gene transcription, the possibility is raised that the transcription-modulatory region directs a transcription initiation event similar to open complex formation at procaryotic promoters.

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

1984 ◽  
Vol 4 (12) ◽  
pp. 2876-2882 ◽  
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.

Sequential transcription-translation of simian virus 40 by using mammalian cell extracts

1981 ◽  
Vol 1 (10) ◽  
pp. 919-931
Author(s):  
C L Cepko ◽  
U Hansen ◽  
H Handa ◽  
P A Sharp
Keyword(s):  
Transcription Initiation ◽  
Simian Virus 40 ◽  
Restriction Enzymes ◽  
Simian Virus ◽  
T Antigen ◽  
Coding Regions ◽  
Cell Extracts ◽  
Initiation Of Translation

Ribonucleic acids (RNAs) transcribed in vitro by using the whole-cell extract system of Manley et al. (Proc. Natl. Acad. Sci. U.S.A. 77:3855-3859, 1980) were tested for their efficiency and fidelity in directing protein synthesis in reticulocyte lysates. Simian virus 40 deoxyribonucleic acid (DNA), cleaved by various restriction endonucleases, was used as the template. Successful translation of the small tumor antigen t, as well as the capsid proteins VP1, VP2, and VP3, was detected by immunoprecipitation analysis. Although no synthesis of large T antigen was detected, use of this technology allows detection of large T synthesis resulting from the correct splicing of as little as 0.2% of the in vitro RNA transcripts, making it ideal for use as an in vitro splicing assay. Transcripts synthesized in vitro were used as messages at least as efficiently as were viral messenger RNA's (mRNA's) synthesized in vivo; and in the case of small t, there was more efficient translation of small t mRNA synthesized in vitro than of small t mRNA synthesized in vivo. The transcripts that served as mRNA's for the various polypeptides were identified by using the following two criteria. (i) The sensitivity of synthesis of a given protein to digestion of the template DNA with restriction enzymes allowed the localization of the promoter and coding regions. (ii) Translation of size-fractionated RNA allowed confirmation of the transcript-mRNA assignments. With these techniques we found that VP2, VP3 and, in some cases, VP1 synthesis resulted from the initiation of translation at internal AUG codons. In fact, families of polypeptides were produced by initiation of translation at AUG codons within sequences coding for VP1 and T, presumably as a result of transcription initiation events that generated 5' ends immediately upstream from these AUGs. Application of this technology for the identification of coding regions within cloned DNA fragments is discussed.

THE IN VITRO TRANSCRIPTION OF DROSOPHILA tRNA GENES

1982 ◽  
pp. 219-234
Author(s):  
T. Dingermann ◽  
S. Sharp ◽  
J. Schaack ◽  
D. DeFranco ◽  
D.L. Johnson ◽  
...  
Keyword(s):  
In Vitro Transcription ◽  
Trna Genes
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