Architecture and folding mechanism of the Azoarcus Group I Pre-tRNA

Rangan, Prashanth; Masquida, Benoı̂t; Westhof, Éric; Woodson, Sarah A.

doi:10.1016/j.jmb.2004.03.059

Cited by 44 publications

(32 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Studies of tRNA folding and stability have benefited from the development of a variety of techniques, including high-resolution footprinting and single-molecule FRET (fluorescence resonance energy transfer) to study equilibrium structures (Chakshusmathi et al 2003;Rangan et al 2004;Wang et al 2008;Messmer et al 2009;Dammertz et al 2011), nuclear magnetic resonance to provide shortrange dynamics information (Vermeulen et al 2005;Farjon et al 2009), and birefringence decay and phased t ratio analysis to address long-range flexibility (Friederich et al 1998). The folding kinetics of fluorescently labeled tRNA has been followed directly (Serebrov et al 2001).…”

Section: Introductionmentioning

confidence: 99%

Kinetics of tRNA folding monitored by aminoacylation

Bhaskaran

Rodríguez-Hernández²,

Perona³

2012

RNA

View full text Add to dashboard Cite

We describe a strategy for tracking Mg 2+ -initiated folding of 32 P-labeled tRNA molecules to their native structures based on the capacity for aminoacylation by the cognate aminoacyl-tRNA synthetase enzyme. The approach directly links folding to function, paralleling a common strategy used to study the folding of catalytic RNAs. Incubation of unfolded tRNA with magnesium ions, followed by the addition of aminoacyl-tRNA synthetase and further incubation, yields a rapid burst of aminoacyl-tRNA formation corresponding to the prefolded tRNA fraction. A subsequent slower increase in product formation monitors continued folding in the presence of the enzyme. Further analysis reveals the presence of a parallel fraction of tRNA that folds more rapidly than the majority of the population. The application of the approach to study the influence of post-transcriptional modifications in folding of Escherichia coli tRNA 1 Gln reveals that the modified bases increase the folding rate but do not affect either the equilibrium between properly folded and misfolded states or the folding pathway. This assay allows the use of 32 P-labeled tRNA in integrated studies combining folding, post-transcriptional processing, and aminoacylation reactions.

show abstract

Section: Introductionmentioning

confidence: 99%

Kinetics of tRNA folding monitored by aminoacylation

Bhaskaran

Rodríguez-Hernández²,

Perona³

2012

RNA

View full text Add to dashboard Cite

show abstract

“…1). Base pairing of the substrate RNA to a guide sequence in the ribozyme produces the P1 splice site helix, which docks into the active site via tertiary interactions with the core of the folded ribozyme (9,10). An exogenous G nucleotide binds a pocket in P7, from which its 3Ј-OH attacks the splice site phosphodiester, cleaving the RNA substrate into two shorter fragments (9 -12).…”

mentioning

confidence: 99%

Increased Ribozyme Activity in Crowded Solutions

Desai

Kilburn

Lee

et al. 2014

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…5A; ReinholdHurek and Shub 1992; Adams et al 2004a;Rangan et al 2004). This anticodon stem contains five base pairs between the 5 ′ exon and the 3 ′ exon, 3 nt distant from the 5 ′ and 3 ′ splice site, respectively.…”

Section: Internalmentioning

confidence: 99%

“…Ile sequence (Rangan et al 2004) at the flanks of the Azoarcus ribozyme, thereby replacing the corresponding CAT sequence (Fig. 7, construct CisAzoCAT_tRNA).…”

Section: In Vivo Activity Of the Azoarcus Ribozyme In E Coli Cellsmentioning

confidence: 99%

“…1A,B). The Azoarcus ribozyme folds into its active conformation faster than the Tetrahymena ribozyme in vitro (Russell and Herschlag 2001;Rangan et al 2004;Mitra et al 2011;Sinan et al 2011), and it can mediate trans-ligation reactions at high concentrations of magnesium, ribozyme, and at elevated temperature (Chowrira et al 1993;Hayden and Lehman 2006;Burton and Lehman 2010). The structure of the Azoarcus ribozyme is known at the highest detail of all group I intron ribozymes, for different stages of the splicing pathway (Adams et al 2004a,b;Lipchock and Strobel 2008).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Trans-splicing with the group I intron ribozyme from Azoarcus

Dolan

Müller

2013

RNA

View full text Add to dashboard Cite

Group I introns are ribozymes (catalytic RNAs) that excise themselves from RNA primary transcripts by catalyzing two successive transesterification reactions. These cis-splicing ribozymes can be converted into trans-splicing ribozymes, which can modify the sequence of a separate substrate RNA, both in vitro and in vivo. Previous work on trans-splicing ribozymes has mostly focused on the 16S rRNA group I intron ribozyme from Tetrahymena thermophila. Here, we test the trans-splicing potential of the tRNA Ile group I intron ribozyme from the bacterium Azoarcus. This ribozyme is only half the size of the Tetrahymena ribozyme and folds faster into its active conformation in vitro. Our results showed that in vitro, the Azoarcus and Tetrahymena ribozymes favored the same set of splice sites on a substrate RNA. Both ribozymes showed the same trans-splicing efficiency when containing their individually optimized 5 ′ terminus. In contrast to the previously optimized 5 ′ -terminal design of the Tetrahymena ribozyme, the Azoarcus ribozyme was most efficient with a trans-splicing design that resembled the secondary structure context of the natural cis-splicing Azoarcus ribozyme, which includes base-pairing between the substrate 5 ′ portion and the ribozyme 3 ′ exon. These results suggested preferred trans-splicing interactions for the Azoarcus ribozyme under near-physiological in vitro conditions. Despite the high activity in vitro, however, the splicing efficiency of the Azoarcus ribozyme in Escherichia coli cells was significantly below that of the Tetrahymena ribozyme.

show abstract

Architecture and folding mechanism of the Azoarcus Group I Pre-tRNA

Cited by 44 publications

References 45 publications

Kinetics of tRNA folding monitored by aminoacylation

Kinetics of tRNA folding monitored by aminoacylation

Increased Ribozyme Activity in Crowded Solutions

Trans-splicing with the group I intron ribozyme from Azoarcus

Contact Info

Product

Resources

About