The use of a synthetic tRNA gene as a novel approach to study<i>in vivo</i>transcription and chromatin structure in yeast

Krieg, Robert; Stucka, Rolf; Clark, Shawna; Feldmann, Horst

doi:10.1093/nar/19.14.3849

Cited by 13 publications

(8 citation statements)

References 42 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Northern hybridization analysis with endogenous, intronless tRNA Glu (TTC) as an internal standard (43). We have previously analyzed the in vivo expression of two gene fusions, called [5ЈCR]Syn2 and [5ЈNR]Syn2, in which the 5Ј-flanking region of either N(GTT)CR or N(GTT)NR, a member of the tRNA Asn (GTT) gene family, is fused to tDNA Syn2 .…”

Section: Resultsmentioning

confidence: 99%

Requirement of Nhp6 Proteins for Transcription of a Subset of tRNA Genes and Heterochromatin Barrier Function in Saccharomyces cerevisiae

Braglia

Dugas

Donze

et al. 2007

Molecular and Cellular Biology

View full text Add to dashboard Cite

A key event in tRNA gene (tDNA) transcription by RNA polymerase (Pol) III is the TFIIIC-dependent assembly of TFIIIB upstream of the transcription start site. Different tDNA upstream sequences bind TFIIIB with different affinities, thereby modulating tDNA transcription. We found that in the absence of Nhp6 proteins, the influence of the 5-flanking region on tRNA gene transcription is dramatically enhanced in Saccharomyces cerevisiae. Expression of a tDNA bearing a suboptimal TFIIIB binding site, but not of a tDNA preceded by a strong TFIIIB binding region, was strongly dependent on Nhp6 in vivo. Upstream sequencedependent stimulation of tRNA gene transcription by Nhp6 could be reproduced in vitro, and Nhp6 proteins were found associated with tRNA genes in yeast cells. We also show that both transcription and silencing barrier activity of a tDNA Thr at the HMR locus are compromised in the absence of Nhp6. Our data suggest that Nhp6 proteins are important components of Pol III chromatin templates that contribute both to the robustness of tRNA gene expression and to positional effects of Pol III transcription complexes.Transcription of tRNA genes (tDNAs) by RNA polymerase (Pol) III in yeast involves multistep assembly of transcription factors into a preinitiation complex that recruits Pol III (14, 27). Two highly conserved internal control regions, the A and B blocks, together form a specific binding site for multisubunit transcription factor IIIC (TFIIIC). Promoter-bound TFIIIC provides an interaction platform for the productive assembly of TFIIIB in an ϳ50-bp region upstream of the transcription start site (TSS). The TFIIIB-DNA complex is by itself capable of productively recruiting Pol III and supporting multiple rounds of transcription in vitro (36). Transcription then proceeds through a facilitated reinitiation pathway that involves Pol recapture after transcription termination (19,20,25). Accumulating evidence suggests that the transcription complexes assembled on class III genes may positionally influence other genomic transactions, such as Ty element retroposition (2, 16, 40) and the expression of neighboring genes. In the latter case, tDNAs can act as repressor elements (9, 34) or as barriers to the spread of silencing (22, 59). Despite the remarkable stability of the TFIIIB-DNA complex and its centrality in the transcription mechanism, TFIIIB contacts with its upstream DNA binding region are not based on simple sequence specificity rules. The 5Ј-flanking region of tRNA genes has long been known to modulate the efficiency of tRNA gene transcription (61). We have recently shown that the transcriptional strength of tRNA gene upstream regions correlates, at least in yeast, with the occurrence of a composite sequence pattern within the TFIIIB binding region and that degenerated yet recognizable sequence patterns also occur upstream of tDNAs in many eukaryotic genomes (29). Yeast tDNA upstream regions also display a remarkable bending propensity, a feature that might facilitate TFIIIB binding (29,30). In Saccha...

show abstract

Section: Resultsmentioning

confidence: 99%

Requirement of Nhp6 Proteins for Transcription of a Subset of tRNA Genes and Heterochromatin Barrier Function in Saccharomyces cerevisiae

Braglia

Dugas

Donze

et al. 2007

Molecular and Cellular Biology

View full text Add to dashboard Cite

show abstract

“…In principle, this should be possible; many organisms (including plants) contain tRNA genes with introns (generally 10-30 nucleotides in length and with few sequence constraints) which appear to have little effect on transcription or on the tertiary structure of the transcript (e.g. Lee and Knapp, 1985;Stange et al, 1988 (Krieg et al, 1991). Although considerable progress is clearly necessary before this becomes a practical means of influencing mitochondrial gene expression, given the current difficulties with direct transformation of mitochondria (it has yet to be achieved with higher plant mitochondria), this alternative approach seems to be worth pursuing.…”

Section: Discussionmentioning

confidence: 99%

In vivo import of a normal or mutagenized heterologous transfer RNA into the mitochondria of transgenic plants: towards novel ways of influencing mitochondrial gene expression?

1992

Trends in Genetics

View full text Add to dashboard Cite

Evidence that nuclear-encoded RNAs are present inside mitochondria has been reported from a wide variety of organisms, and is presumed to be due to import of specific cytosolic RNAs. In plants, the first examples were the mitochondrial leucine transfer RNAs of bean. In all cases, the evidence is circumstantial, based on hybridization of the mitochondrial RNAs to nuclear and not mitochondrial DNA. Here we show that transgenic potato plants carrying a leucine tRNA gene from bean nuclear DNA contain RNA transcribed from the introduced gene both in the cytosol and inside mitochondria, providing proof that the mitochondrial leucine tRNA is derived from a nuclear gene and imported into the mitochondria. The same bean gene carrying a 4 bp insertion in the anticodon loop was also expressed in transgenic potato plants and the transcript found to be present inside mitochondria, suggesting that this natural RNA import system could eventually be used to introduce foreign RNA sequences into mitochondria.

show abstract

“…Mutant tRNAs or tRNA mimics can be generated either by in vitro transcription (see for instance [54,55]) or in vivo expression [56][57][58] of mutated tRNA genes. Because tRNA has a complex tertiary structure [59,60], some mutations and modifications will inevitably lead to misfolding [61].…”

Section: Acceptable Limits Of Trna Mutagenesismentioning

confidence: 99%

Misacylation of tRNA in prokaryotes: a re-evaluation

Stortchevoi

2006

Cell. Mol. Life Sci.

View full text Add to dashboard Cite

Misacylation of tRNA by a non-cognate amino acid is a natural phenomenon and occurs with a frequency of approximately 1 in 10,000 due to occasional mistakes in aminoacyl transfer RNA (tRNA) synthesis. In a number of prokaryotic organisms, misacylation of selenocysteinyl tRNA, glutaminyl tRNA and aspartyl tRNAs has particular physiological meaning. Recently, misacylation has emerged as a powerful tool for studying specific interactions between aa-tRNAs and associated protein factors. The present review provides an overview of the application of misacylated tRNA in research.

show abstract

The use of a synthetic tRNA gene as a novel approach to studyin vivotranscription and chromatin structure in yeast

Cited by 13 publications

References 42 publications

Requirement of Nhp6 Proteins for Transcription of a Subset of tRNA Genes and Heterochromatin Barrier Function in Saccharomyces cerevisiae

Requirement of Nhp6 Proteins for Transcription of a Subset of tRNA Genes and Heterochromatin Barrier Function in Saccharomyces cerevisiae

In vivo import of a normal or mutagenized heterologous transfer RNA into the mitochondria of transgenic plants: towards novel ways of influencing mitochondrial gene expression?

Misacylation of tRNA in prokaryotes: a re-evaluation

Contact Info

Product

Resources

About