Improved method for PCR-mediated site-directed mutagenesis

Barettino, Domingo; Feigenbutz, Monika; Valcárcel, Rafael; Stunnenberg, Hendrik G.

doi:10.1093/nar/22.3.541

Cited by 116 publications

(76 citation statements)

References 6 publications

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“…35 using the following primers: Ϫ20 primer, 5Ј-GTAAAACGACGGCCAGT; EcoRI clockwise, 5Ј-GTATCACGAGGCCCTTTCG; U21A, 5Ј-CACGT-CATGTGGCAGAAAGCC; and A9U, 5Ј-GGCAGAAAGCCACAAGCGC-CGGGCAC (mutant nucleotides are printed in bold face).…”

Section: Methodsmentioning

confidence: 99%

Temporal Translational Control by a Metastable RNA Structure

Møller‐Jensen

Franch

Gerdes

2001

Journal of Biological Chemistry

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Programmed cell death by the hok/sok locus of plasmid R1 relies on a complex translational control mechanism. The highly stable hok mRNA is activated by 3-end exonucleolytical processing. Removal of the mRNA 3 end releases a 5-end sequence that triggers refolding of the mRNA. The refolded hok mRNA is translatable but can also bind the inhibitory Sok antisense RNA. Binding of Sok RNA leads to irreversible mRNA inactivation by an RNase III-dependent mechanism. A coherent model predicts that during transcription hok mRNA must be refractory to translation and antisense RNA binding. Here we provide genetic evidence for the existence of a 5 metastable structure in hok mRNA that locks the nascent transcript in an inactive configuration in vivo. Consistently, the metastable structure reduces the rate of Sok RNA binding and completely blocks hok translation in vitro. Structural analyses of native RNAs strongly support that the 5 metastable structure exists in the nascent transcript. Further structural analyses reveal that the mRNA 3 end triggers refolding of the mRNA 5 end into the more stable tac-stem conformation. These results provide a profound understanding of an unusual and intricate posttranscriptional control mechanism.RNA molecules fold into highly ordered structures essential to their diverse biological functions. Accordingly, the question of how the linear sequence of ribonucleotides dictates the overall folding of an RNA has received much attention (1-6). Folding of RNA, whether occurring from a denatured state or sequentially in concomitance with its synthesis, involves the formation of a number of hierarchically ordered intramolecular interactions leading to increasing levels of structural organization. In both cases, structural rearrangements of kinetically favored folding intermediates may occur before the thermodynamically most stable conformation is reached (7,8). In the course of folding, RNA molecules face the risk of being trapped in nonequilibrium conformations. Because of the high thermodynamic stability of RNA secondary structures, rearrangement of such nonequilibrium conformations can constitute a substantial energy barrier, thus leading to kinetic trapping of the RNA in thermodynamically suboptimal conformations termed metastable structures (9 -11). Metastable folding intermediates have proved important to a number of biological processes including plasmid replication (12), replication of RNA by Q␤ replicase (13, 14), human immunodeficiency virus-1 RNA export to the cytoplasm (15), ribozyme activity (16 -20), and viroid replication (21-23).The hok/sok locus of plasmid R1 mediates plasmid maintenance by the killing of plasmid-free cells, also termed postsegregational killing (PSK) 1 (24). The PSK mechanism, which restricts synthesis of Hok toxin to newborn plasmid-free cells, is controlled entirely at the post-transcriptional level. The hok/ sok locus, presented schematically in Fig. 1, specifies two transcripts: the toxin-encoding hok (host killing) mRNA and the labile antisense inhibitor Sok RN...

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Section: Methodsmentioning

confidence: 99%

Temporal Translational Control by a Metastable RNA Structure

Møller‐Jensen

Franch

Gerdes

2001

Journal of Biological Chemistry

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“…Site-directed mutagenesis of xopD was performed using the PCRmediated megaprimer method (Barettino et al, 1994). In the first PCR amplification, pEG202-nls(xopD) was used as a template, and MB160 primer and individual mutagenic primers were used (see Supplemental Table 2 online).…”

Section: Xopd Mutagenesismentioning

confidence: 99%

XopD SUMO Protease Affects Host Transcription, Promotes Pathogen Growth, and Delays Symptom Development inXanthomonas-Infected Tomato Leaves

et al. 2008

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We demonstrate that XopD, a type III effector from Xanthomonas campestris pathovar vesicatoria (Xcv), suppresses symptom production during the late stages of infection in susceptible tomato (Solanum lycopersicum) leaves. XopD-dependent delay of tissue degeneration correlates with reduced chlorophyll loss, reduced salicylic acid levels, and changes in the mRNA abundance of senescence- and defense-associated genes despite high pathogen titers. Subsequent structure-function analyses led to the discovery that XopD is a DNA binding protein that alters host transcription. XopD contains a putative helix-loop-helix domain required for DNA binding and two conserved ERF-associated amphiphilic motifs required to repress salicylic acid– and jasmonic acid–induced gene transcription in planta. Taken together, these data reveal that XopD is a unique virulence factor in Xcv that alters host transcription, promotes pathogen multiplication, and delays the onset of leaf chlorosis and necrosis.

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“…Construct UP-GG was derived from UP-GG+100 by deleting the 54 bp SalI fragment from the duplicated polylinkers. Construct UP-GA was derived from UP-GG by site-directed mutagenesis (64). All constructs were verified by sequencing.…”

Section: Oligonucleotides and Plasmidsmentioning

confidence: 99%

Sequences upstream of the branch site are required to form helix II between U2 and U6 snRNA in a trans-splicing reaction

Ast

Pavelitz

Weiner

2001

Nucleic Acids Research

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Three different base paired stems form between U2 and U6 snRNA over the course of the mRNA splicing reaction (helices I, II and III). One possible function of U2/U6 helix II is to facilitate subsequent U2/U6 helix I and III interactions, which participate directly in catalysis. Using an in vitro trans-splicing assay, we investigated the function of sequences located just upstream from the branch site (BS). We find that these upstream sequences are essential for stable binding of U2 to the branch region, and for U2/U6 helix II formation, but not for initial U2/BS pairing. We also show that non-functional upstream sequences cause U2 snRNA stem-loop IIa to be exposed to dimethylsulfate modification, perhaps reflecting a U2 snRNA conformational change and/or loss of SF3b proteins. Our data suggest that initial binding of U2 snRNP to the BS region must be stabilized by an interaction with upstream sequences before U2/U6 helix II can form or U2 stem-loop IIa can participate in spliceosome assembly.

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Improved method for PCR-mediated site-directed mutagenesis

Cited by 116 publications

References 6 publications

Temporal Translational Control by a Metastable RNA Structure

Temporal Translational Control by a Metastable RNA Structure

XopD SUMO Protease Affects Host Transcription, Promotes Pathogen Growth, and Delays Symptom Development inXanthomonas-Infected Tomato Leaves

Sequences upstream of the branch site are required to form helix II between U2 and U6 snRNA in a trans-splicing reaction

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