5′-flanking sequences required for efficient transcription in vitro of 5S RNA genes, in the related nematodes Caenorhabditis elegans and Caenorhabditis briggsae

An extract from whole oocytes of Xenopus laevis was shown to transcribe somatic-type 5S RNA genes approximately 100-fold more efficiently than oocyte-type 5S RNA genes. This preference was at least 10-fold greater than the preference seen upon microinjection of 5S RNA genes into oocyte nuclei or upon in vitro transcription in an oocyte nuclear extract. The approximately 100-fold transcriptional bias in favor of the somatic-type 5S RNA genes observed in vitro in the whole oocyte extract was similar to the transcriptional bias observed in developing Xenopus embryos. We also showed that in the whole oocyte extract, a promoter-binding protein required for 5S RNA gene transcription, TFIIIA, was bound both to the actively transcribed somatic-type 5S RNA gene and to the largely inactive oocyte-type 5S RNA genes. These findings suggest that the mechanism for the differential expression of 5S RNA genes during Xenopus development does not involve differential binding of TFIIIA to 5S RNA genes.

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Transcriptionally inactive oocyte-type 5S RNA genes of Xenopus laevis are complexed with TFIIIA in vitro

Molecular and Cellular Biology ◽

10.1128/mcb.7.10.3503-3510.1987 ◽

1987 ◽

Vol 7 (10) ◽

pp. 3503-3510

Author(s):

L J Peck ◽

L Millstein ◽

P Eversole-Cire ◽

J M Gottesfeld ◽

A Varshavsky

Keyword(s):

Xenopus Laevis ◽

Differential Expression ◽

Gene Transcription ◽

Nuclear Extract ◽

In Vitro Transcription ◽

5S Rna ◽

Differential Binding ◽

5S Rna Genes ◽

Rna Genes

An extract from whole oocytes of Xenopus laevis was shown to transcribe somatic-type 5S RNA genes approximately 100-fold more efficiently than oocyte-type 5S RNA genes. This preference was at least 10-fold greater than the preference seen upon microinjection of 5S RNA genes into oocyte nuclei or upon in vitro transcription in an oocyte nuclear extract. The approximately 100-fold transcriptional bias in favor of the somatic-type 5S RNA genes observed in vitro in the whole oocyte extract was similar to the transcriptional bias observed in developing Xenopus embryos. We also showed that in the whole oocyte extract, a promoter-binding protein required for 5S RNA gene transcription, TFIIIA, was bound both to the actively transcribed somatic-type 5S RNA gene and to the largely inactive oocyte-type 5S RNA genes. These findings suggest that the mechanism for the differential expression of 5S RNA genes during Xenopus development does not involve differential binding of TFIIIA to 5S RNA genes.

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An upstream signal is required for in vitro transcription ofNeurospora 5S RNA genes

MGG Molecular & General Genetics ◽

10.1007/bf02428052 ◽

1986 ◽

Vol 205 (1) ◽

pp. 189-192 ◽

Cited By ~ 26

Author(s):

Eric U. Selker ◽

Ewa Morzycka-Wroblewska ◽

Judith N. Stevens ◽

Robert L. Metzenberg

Keyword(s):

In Vitro Transcription ◽

5S Rna ◽

5S Rna Genes ◽

Rna Genes

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Selective in vitro transcription of the 5S RNA genes of a DNA template

Biochemistry ◽

10.1021/bi00582a008 ◽

1979 ◽

Vol 18 (15) ◽

pp. 3243-3248 ◽

Cited By ~ 6

Author(s):

Steven Ackerman ◽

John J. Furth

Keyword(s):

In Vitro Transcription ◽

5S Rna ◽

Dna Template ◽

5S Rna Genes ◽

Rna Genes

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Role of TFIIIA zinc fingers in vivo: analysis of single-finger function in developing Xenopus embryos.

Molecular and Cellular Biology ◽

10.1128/mcb.13.8.4776 ◽

1993 ◽

Vol 13 (8) ◽

pp. 4776-4783 ◽

Cited By ~ 19

Author(s):

M B Rollins ◽

S Del Rio ◽

A L Galey ◽

D R Setzer ◽

M T Andrews

Keyword(s):

Transcription Factor ◽

Zinc Finger ◽

Transcriptional Activation ◽

Zinc Fingers ◽

Wild Type ◽

5S Rna ◽

5S Rna Genes ◽

Rna Genes

The Xenopus 5S RNA gene-specific transcription factor IIIA (TFIIIA) has nine consecutive Cys2His2 zinc finger motifs. Studies were conducted in vivo to determine the contribution of each of the nine zinc fingers to the activity of TFIIIA in living cells. Nine separate TFIIIA mutants were expressed in Xenopus embryos following microinjection of their respective in vitro-derived mRNAs. Each mutant contained a single histidine-to-asparagine substitution in the third zinc ligand position of an individual zinc finger. These mutations result in structural disruption of the mutated finger with little or no effect on the other fingers. The activity of mutant proteins in vivo was assessed by measuring transcriptional activation of the endogenous 5S RNA genes. Mutants containing a substitution in zinc finger 1, 2, or 3 activate 5S RNA genes at a level which is reduced relative to that in embryos injected with the message for wild-type TFIIIA. Proteins with a histidine-to-asparagine substitution in zinc finger 5 or 7 activate 5S RNA genes at a level that is roughly equivalent to that of the wild-type protein. Zinc fingers 8 and 9 appear to be critical for the normal function of TFIIIA, since mutations in these fingers result in little or no activation of the endogenous 5S RNA genes. Surprisingly, proteins with a mutation in zinc finger 4 or 6 stimulate 5S RNA transcription at a level that is significantly higher than that mediated by similar concentrations of wild-type TFIIIA. Differences in the amount of newly synthesized 5S RNA in embryos containing the various mutant forms of TFIIIA result from differences in the relative number and/or activity of transcription complexes assembled on the endogenous 5S RNA genes and, in the case of the finger 4 and finger 6 mutants, result from increased transcriptional activation of the normally inactive oocyte-type 5S RNA genes. The remarkably high activity of the finger 6 mutant can be reproduced in vitro when transcription is carried out in the presence of 5S RNA. Disruption of zinc finger 6 results in a form of TFIIIA that exhibits reduced susceptibility to feedback inhibition by 5S RNA and therefore increases the availability of the transcription factor for transcription complex formation.

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Role of TFIIIA zinc fingers in vivo: analysis of single-finger function in developing Xenopus embryos

Molecular and Cellular Biology ◽

10.1128/mcb.13.8.4776-4783.1993 ◽

1993 ◽

Vol 13 (8) ◽

pp. 4776-4783

Author(s):

M B Rollins ◽

S Del Rio ◽

A L Galey ◽

D R Setzer ◽

M T Andrews

Keyword(s):

Transcription Factor ◽

Zinc Finger ◽

Transcriptional Activation ◽

Zinc Fingers ◽

Wild Type ◽

5S Rna ◽

5S Rna Genes ◽

Rna Genes

The Xenopus 5S RNA gene-specific transcription factor IIIA (TFIIIA) has nine consecutive Cys2His2 zinc finger motifs. Studies were conducted in vivo to determine the contribution of each of the nine zinc fingers to the activity of TFIIIA in living cells. Nine separate TFIIIA mutants were expressed in Xenopus embryos following microinjection of their respective in vitro-derived mRNAs. Each mutant contained a single histidine-to-asparagine substitution in the third zinc ligand position of an individual zinc finger. These mutations result in structural disruption of the mutated finger with little or no effect on the other fingers. The activity of mutant proteins in vivo was assessed by measuring transcriptional activation of the endogenous 5S RNA genes. Mutants containing a substitution in zinc finger 1, 2, or 3 activate 5S RNA genes at a level which is reduced relative to that in embryos injected with the message for wild-type TFIIIA. Proteins with a histidine-to-asparagine substitution in zinc finger 5 or 7 activate 5S RNA genes at a level that is roughly equivalent to that of the wild-type protein. Zinc fingers 8 and 9 appear to be critical for the normal function of TFIIIA, since mutations in these fingers result in little or no activation of the endogenous 5S RNA genes. Surprisingly, proteins with a mutation in zinc finger 4 or 6 stimulate 5S RNA transcription at a level that is significantly higher than that mediated by similar concentrations of wild-type TFIIIA. Differences in the amount of newly synthesized 5S RNA in embryos containing the various mutant forms of TFIIIA result from differences in the relative number and/or activity of transcription complexes assembled on the endogenous 5S RNA genes and, in the case of the finger 4 and finger 6 mutants, result from increased transcriptional activation of the normally inactive oocyte-type 5S RNA genes. The remarkably high activity of the finger 6 mutant can be reproduced in vitro when transcription is carried out in the presence of 5S RNA. Disruption of zinc finger 6 results in a form of TFIIIA that exhibits reduced susceptibility to feedback inhibition by 5S RNA and therefore increases the availability of the transcription factor for transcription complex formation.

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A quantitative assay for Xenopus 5S RNA gene transcription in vitro

Cell ◽

10.1016/0092-8674(81)90106-9 ◽

1981 ◽

Vol 24 (3) ◽

pp. 809-817 ◽

Cited By ~ 62

Author(s):

W.Michael Wormington ◽

Daniel F. Bogenhagen ◽

Eddie Jordan ◽

Donald D. Brown

Keyword(s):

Gene Transcription ◽

Quantitative Assay ◽

5S Rna ◽

Transcription In Vitro

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Regular arrangement of nucleosomes on 5S rRNA genes in Xenopus laevis

Molecular and Cellular Biology ◽

10.1128/mcb.3.4.720-730.1983 ◽

1983 ◽

Vol 3 (4) ◽

pp. 720-730

Author(s):

D Young ◽

D Carroll

Keyword(s):

Xenopus Laevis ◽

Chromatin Structure ◽

Micrococcal Nuclease ◽

Rrna Genes ◽

Dna Repeats ◽

5S Rna ◽

5S Dna ◽

Regular Arrangement ◽

5S Rna Genes ◽

Rna Genes

The chromatin structure of the oocyte-type 5S RNA genes in Xenopus laevis was investigated. Blot hybridization analysis of DNA from micrococcal nuclease digests of erythrocyte nuclei showed that 5S DNA has the same average nucleosome repeat length, 192 +/- 4 base pairs, as two Xenopus satellite DNAs and bulk erythrocyte chromatin. The positions of nuclease-sensitive regions in the 5S DNA repeats of purified DNA and chromatin from erythrocytes were mapped by using an indirect end-labeling technique. Although most of the sites cleaved in purified DNA were also cleaved in chromatin, the patterns of intensities were strikingly different in the two cases. In 5S chromatin, three nuclease-sensitive regions were spaced approximately a nucleosome length apart, suggesting a single, regular arrangement of nucleosomes on most of the 5S DNA repeats. The observed nucleosome locations are discussed with respect to nucleotide sequences known to be important for expression of 5S RNA. Because the preferred locations appear to be reestablished in each repeating unit, despite spacer length heterogeneity, we suggest that the regular chromatin structure reflects the presence of a sequence-specific DNA-binding component on inactive 5S RNA genes.

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