La Protein and the Trafficking of Nascent RNA Polymerase III Transcripts

Xenopus RNA polymerase III specifically initiates transcription on poly(dC)-tailed DNA templates in the absence of other class III transcription factors normally required for transcription initiation. In experimental analyses of transcription termination using DNA fragments with a 5S rRNA gene positioned downstream of the tailed end, only 40% of the transcribing polymerase molecules terminate at the normally efficient Xenopus borealis somatic-type 5S rRNA terminators; the remaining 60% read through these signals and give rise to runoff transcripts. We find that the nascent RNA strand is inefficiently displaced from the DNA template during transcription elongation. Interestingly, only polymerases synthesizing a displaced RNA terminate at the 5S rRNA gene terminators; when the nascent RNA is not displaced from the template, read-through transcripts are synthesized. RNAs with 3' ends at the 5S rRNA gene terminators are judged to result from authentic termination events on the basis of multiple criteria, including kinetic properties, the precise 3' ends generated, release of transcripts from the template, and recycling of the polymerase. Even though only 40% of the polymerase molecules ultimately terminate at either of the tandem 5S rRNA gene terminators, virtually all polymerases pause there, demonstrating that termination signal recognition can be experimentally uncoupled from polymerase release. Thus, termination is dependent on RNA strand displacement during transcription elongation, whereas termination signal recognition is not. We interpret our results in terms of a two-step model for transcription termination in which polymerase release is dependent on the fate of the nascent RNA strand during transcription elongation.

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Mutations in the RNA Polymerase III Subunit Rpc11p That Decrease RNA 3′ Cleavage Activity Increase 3′-Terminal Oligo(U) Length and La-Dependent tRNA Processing

Molecular and Cellular Biology ◽

10.1128/mcb.25.2.621-636.2005 ◽

2005 ◽

Vol 25 (2) ◽

pp. 621-636 ◽

Cited By ~ 40

Author(s):

Ying Huang ◽

Robert V. Intine ◽

Amy Mozlin ◽

Samuel Hasson ◽

Richard J. Maraia

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Iii ◽

Protein A ◽

Activity Increase ◽

Cleavage Activity ◽

Polymerase Iii ◽

La Protein ◽

Pol Iii ◽

And Function ◽

Zinc Ribbon

ABSTRACT Termination by RNA polymerase III (Pol III) produces RNAs whose 3′ oligo(U) termini are bound by La protein, a chaperone that protects RNAs from 3′ exonucleases and promotes their maturation. Multiple reports indicate that yeasts use La-dependent and -independent pathways for tRNA maturation, with defective pre-tRNAs being most sensitive to decay and most dependent on La for maturation and function. The Rpc11p subunit of Pol III shows homology with the zinc ribbon of TFIIS and is known to mediate RNA 3′ cleavage and to be important for termination. We used a La-dependent opal suppressor, tRNASerUGAM, which suppresses ade6-704 and the accumulation of red pigment, to screen Schizosaccaromyces pombe for rpc11 mutants that increase tRNA-mediated suppression. Analyses of two zinc ribbon mutants indicate that they are deficient in Pol III RNA 3′ cleavage activity and produce pre-tRNASerUGAM transcripts with elongated 3′-oligo(U) tracts that are better substrates for La. A substantial fraction of pre-tRNASerUGAM contains too few 3′ Us for efficient La binding and appears to decay in wild-type cells but has elongated oligo(U) tracts and matures along the La-dependent pathway in the mutants. The data indicate that Rpc11p limits RNA 3′-U length and that this significantly restricts pre-tRNAs to a La-independent pathway of maturation in fission yeast.

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Ro small cytoplasmic ribonucleoproteins are a subclass of La ribonucleoproteins: further characterization of the Ro and La small ribonucleoproteins from uninfected mammalian cells

Molecular and Cellular Biology ◽

10.1128/mcb.1.12.1138-1149.1981 ◽

1981 ◽

Vol 1 (12) ◽

pp. 1138-1149

Author(s):

J P Hendrick ◽

S L Wolin ◽

J Rinke ◽

M R Lerner ◽

J A Steitz

Keyword(s):

Rna Polymerase ◽

Mammalian Cells ◽

Protein Complexes ◽

Rna Polymerase Iii ◽

Protein S ◽

Human Cells ◽

Rna Molecules ◽

Transcription System ◽

Polymerase Iii ◽

La Protein

Small ribonucleic acid (RNA)-protein complexes precipitated by anti-Ro and anti-La antibodies from lupus patients have been examined with emphasis on their RNA components. In both ribonucleoprotein (RNP) classes, the numbers of different RNA molecules and their sequences vary between mouse and human cells. The complex mixtures of La RNAs include two previously sequenced 4.5S RNAs from mouse cells and 5S ribosomal RNA-like molecules from both mouse and human cells. All Ro and La RNAs possess 5-triphosphates. Some La RNAs have internal modifications typical of transfer RNAs. The Ro RNPs are quite stable and are localized by immunofluorescence in the cell cytoplasm, whereas the majority of the La RNPs turn over rapidly and reside in the nucleus. Despite these differences, reconstitution experiments show that the Ro particles carry the La as well as the Ro determinant. Studies using a nuclear transcription system demonstrate that most of the La RNAs are synthesized by RNA polymerase III. The possibility that the La protein(s) functions in the transcription or maturation of all RNA polymerase III transcripts is discussed.

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Identification of a La protein binding site in a RNA polymerase III transcript (4.5 I RNA).

Journal of Biological Chemistry ◽

10.1016/s0021-9258(20)82072-9 ◽

1983 ◽

Vol 258 (13) ◽

pp. 8352-8356

Author(s):

R Reddy ◽

D Henning ◽

E Tan ◽

H Busch

Keyword(s):

Rna Polymerase ◽

Protein Binding ◽

Binding Site ◽

Rna Polymerase Iii ◽

Protein Binding Site ◽

Polymerase Iii ◽

La Protein

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Ro small cytoplasmic ribonucleoproteins are a subclass of La ribonucleoproteins: further characterization of the Ro and La small ribonucleoproteins from uninfected mammalian cells.

Molecular and Cellular Biology ◽

10.1128/mcb.1.12.1138 ◽

1981 ◽

Vol 1 (12) ◽

pp. 1138-1149 ◽

Cited By ~ 240

Author(s):

J P Hendrick ◽

S L Wolin ◽

J Rinke ◽

M R Lerner ◽

J A Steitz

Keyword(s):

Rna Polymerase ◽

Mammalian Cells ◽

Protein Complexes ◽

Rna Polymerase Iii ◽

Protein S ◽

Human Cells ◽

Rna Molecules ◽

Transcription System ◽

Polymerase Iii ◽

La Protein

Small ribonucleic acid (RNA)-protein complexes precipitated by anti-Ro and anti-La antibodies from lupus patients have been examined with emphasis on their RNA components. In both ribonucleoprotein (RNP) classes, the numbers of different RNA molecules and their sequences vary between mouse and human cells. The complex mixtures of La RNAs include two previously sequenced 4.5S RNAs from mouse cells and 5S ribosomal RNA-like molecules from both mouse and human cells. All Ro and La RNAs possess 5-triphosphates. Some La RNAs have internal modifications typical of transfer RNAs. The Ro RNPs are quite stable and are localized by immunofluorescence in the cell cytoplasm, whereas the majority of the La RNPs turn over rapidly and reside in the nucleus. Despite these differences, reconstitution experiments show that the Ro particles carry the La as well as the Ro determinant. Studies using a nuclear transcription system demonstrate that most of the La RNAs are synthesized by RNA polymerase III. The possibility that the La protein(s) functions in the transcription or maturation of all RNA polymerase III transcripts is discussed.

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Transcription termination by RNA polymerase III: uncoupling of polymerase release from termination signal recognition.

Molecular and Cellular Biology ◽

10.1128/mcb.12.5.2260 ◽

1992 ◽

Vol 12 (5) ◽

pp. 2260-2272 ◽

Cited By ~ 42

Author(s):

F E Campbell ◽

D R Setzer

Keyword(s):

Rna Polymerase ◽

Transcription Termination ◽

Transcription Elongation ◽

Rna Polymerase Iii ◽

5S Rrna ◽

Rrna Gene ◽

Signal Recognition ◽

Polymerase Iii ◽

5S Rrna Gene ◽

Nascent Rna

Xenopus RNA polymerase III specifically initiates transcription on poly(dC)-tailed DNA templates in the absence of other class III transcription factors normally required for transcription initiation. In experimental analyses of transcription termination using DNA fragments with a 5S rRNA gene positioned downstream of the tailed end, only 40% of the transcribing polymerase molecules terminate at the normally efficient Xenopus borealis somatic-type 5S rRNA terminators; the remaining 60% read through these signals and give rise to runoff transcripts. We find that the nascent RNA strand is inefficiently displaced from the DNA template during transcription elongation. Interestingly, only polymerases synthesizing a displaced RNA terminate at the 5S rRNA gene terminators; when the nascent RNA is not displaced from the template, read-through transcripts are synthesized. RNAs with 3' ends at the 5S rRNA gene terminators are judged to result from authentic termination events on the basis of multiple criteria, including kinetic properties, the precise 3' ends generated, release of transcripts from the template, and recycling of the polymerase. Even though only 40% of the polymerase molecules ultimately terminate at either of the tandem 5S rRNA gene terminators, virtually all polymerases pause there, demonstrating that termination signal recognition can be experimentally uncoupled from polymerase release. Thus, termination is dependent on RNA strand displacement during transcription elongation, whereas termination signal recognition is not. We interpret our results in terms of a two-step model for transcription termination in which polymerase release is dependent on the fate of the nascent RNA strand during transcription elongation.

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