scholarly journals Expanding the binding specificity for RNA recognition by a PUF domain

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Wei Zhou ◽  
Daniel Melamed ◽  
Gabor Banyai ◽  
Cindy Meyer ◽  
Thomas Tuschl ◽  
...  
Keyword(s):  
Rna Binding ◽  
Amino Acid Substitutions ◽  
Rna Recognition ◽  
Specific Interactions ◽  
Wild Type ◽  
Modular Architecture ◽  
Recognition Sequence ◽  
Puf Domain ◽  
Target Rna ◽  
Type Sequence

AbstractThe ability to design a protein to bind specifically to a target RNA enables numerous applications, with the modular architecture of the PUF domain lending itself to new RNA-binding specificities. For each repeat of the Pumilio-1 PUF domain, we generate a library that contains the 8,000 possible combinations of amino acid substitutions at residues critical for RNA contact. We carry out yeast three-hybrid selections with each library against the RNA recognition sequence for Pumilio-1, with any possible base present at the position recognized by the randomized repeat. We use sequencing to score the binding of each variant, identifying many variants with highly repeat-specific interactions. From these data, we generate an RNA binding code specific to each repeat and base. We use this code to design PUF domains against 16 RNAs, and find that some of these domains recognize RNAs with two, three or four changes from the wild type sequence.

Bespoke RNA recognition by Pumilios

2015 ◽  
Vol 43 (5) ◽  
pp. 801-806 ◽  
Author(s):  
Thomas A. Edwards
Keyword(s):  
Rna Binding ◽  
Consensus Sequence ◽  
Rna Binding Protein ◽  
Rna Recognition ◽  
Binding Mechanism ◽  
Recognition Sequence ◽  
Single Base ◽  
Puf Domain ◽  
Binding Sequence

Pumilio is an RNA-binding protein originally identified in Drosophila, with a Puf domain made up of eight Puf repeats, three helix bundles arranged in a rainbow architecture, where each repeat recognizes a single base of the RNA-binding sequence. The eight-base recognition sequence can therefore be modified simply via mutation of the repeat that recognizes the base to be changed and this is understood in detail via high-resolution crystal structures. The binding mechanism is also altered in a variety of homologues from different species, with bases flipped out from the binding site to regenerate a consensus sequence. Thus Pumilios can be designed with bespoke RNA recognition sequences and can be fused to nucleases, split GFP, etc. as tools in vitro and in cells.

scholarly journals A crucial RNA-binding lysine residue in the Nab3 RRM domain undergoes SET1 and SET3-responsive methylation

2020 ◽  
Vol 48 (6) ◽  
pp. 2897-2911 ◽  
Author(s):  
Kwan Yin Lee ◽  
Anand Chopra ◽  
Giovanni L Burke ◽  
Ziyan Chen ◽  
Jack F Greenblatt ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Recognition ◽  
Rna Sequences ◽  
Rna Recognition Motifs ◽  
Rrm Domain ◽  
Lysine Residues ◽  
Recognition Motifs ◽  
Nascent Transcripts ◽  
Target Rna

Abstract The Nrd1–Nab3–Sen1 (NNS) complex integrates molecular cues to direct termination of noncoding transcription in budding yeast. NNS is positively regulated by histone methylation as well as through Nrd1 binding to the initiating form of RNA PolII. These cues collaborate with Nrd1 and Nab3 binding to target RNA sequences in nascent transcripts through their RRM RNA recognition motifs. In this study, we identify nine lysine residues distributed amongst Nrd1, Nab3 and Sen1 that are methylated, suggesting novel molecular inputs for NNS regulation. We identify mono-methylation of one these residues (Nab3-K363me1) as being partly dependent on the H3K4 methyltransferase, Set1, a known regulator of NNS function. Moreover, the accumulation of Nab3-K363me1 is essentially abolished in strains lacking SET3, a SET domain containing protein that is positively regulated by H3K4 methylation. Nab3-K363 resides within its RRM and physically contacts target RNA. Mutation of Nab3-K363 to arginine (Nab3-K363R) decreases RNA binding of the Nab3 RRM in vitro and causes transcription termination defects and slow growth. These findings identify SET3 as a potential contextual regulator of Nab3 function through its role in methylation of Nab3-K363. Consistent with this hypothesis, we report that SET3 exhibits genetic activation of NAB3 that is observed in a sensitized context.

scholarly journals Drosophila Nanos acts as a molecular clamp that modulates the RNA-binding and repression activities of Pumilio

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Chase A Weidmann ◽  
Chen Qiu ◽  
René M Arvola ◽  
Tzu-Fang Lou ◽  
Jordan Killingsworth ◽  
...  
Keyword(s):  
Regulatory Network ◽  
Translational Regulation ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Recognition ◽  
Recognition Sequence ◽  
Repression Complex ◽  
Neuronal Functions ◽  
Rna Complexes

Collaboration among the multitude of RNA-binding proteins (RBPs) is ubiquitous, yet our understanding of these key regulatory complexes has been limited to single RBPs. We investigated combinatorial translational regulation by Drosophila Pumilio (Pum) and Nanos (Nos), which control development, fertility, and neuronal functions. Our results show how the specificity of one RBP (Pum) is modulated by cooperative RNA recognition with a second RBP (Nos) to synergistically repress mRNAs. Crystal structures of Nos-Pum-RNA complexes reveal that Nos embraces Pum and RNA, contributes sequence-specific contacts, and increases Pum RNA-binding affinity. Nos shifts the recognition sequence and promotes repression complex formation on mRNAs that are not stably bound by Pum alone, explaining the preponderance of sub-optimal Pum sites regulated in vivo. Our results illuminate the molecular mechanism of a regulatory switch controlling crucial gene expression programs, and provide a framework for understanding how the partnering of RBPs evokes changes in binding specificity that underlie regulatory network dynamics.

scholarly journals Substrate Range and Genetic Analysis ofAcinetobacter Vanillate Demethylase

2000 ◽  
Vol 182 (5) ◽  
pp. 1383-1389 ◽  
Author(s):  
Birgit Morawski ◽  
Ana Segura ◽  
L. Nicholas Ornston
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitutions ◽  
Missense Mutations ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Nucleotide Substitutions ◽  
Two Component ◽  
Selection For ◽  
Type Sequence ◽  
Selection Of

ABSTRACT An Acinetobacter sp. genetic screen was used to probe structure-function relationships in vanillate demethylase, a two-component monooxygenase. Mutants with null, leaky, and heat-sensitive phenotypes were isolated. Missense mutations tended to be clustered in specific regions, most of which make known contributions to catalytic activity. The vanillate analogsm-anisate, m-toluate, and 4-hydroxy-3,5-dimethylbenzoate are substrates of the enzyme and weakly inhibit the metabolism of vanillate by wild-typeAcinetobacter bacteria. PCR mutagenesis ofvanAB, followed by selection for strains unable to metabolize vanillate, yielded mutant organisms in which vanillate metabolism is more strongly inhibited by the vanillate analogs. Thus, the procedure opens for investigation amino acid residues that may contribute to the binding of either vanillate or its chemical analogs to wild-type and mutant vanillate demethylases. Selection of phenotypic revertants following PCR mutagenesis gave an indication of the extent to which amino acid substitutions can be tolerated at specified positions. In some cases, only true reversion to the original amino acid was observed. In other examples, a range of amino acid substitutions was tolerated. In one instance, phenotypic reversion failed to produce a protein with the original wild-type sequence. In this example, constraints favoring certain nucleotide substitutions appear to be imposed at the DNA level.

MARKER EFFECTS IN THE GENETIC TRANSDUCTION OF TRYPTOPHAN MUTANTS OF E. COLI

Genetics ◽  
1973 ◽  
Vol 75 (3) ◽  
pp. 423-439
Author(s):  
David Stadler ◽  
Beverly Kariya
Keyword(s):  
Low Frequency ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Molecular Configuration ◽  
High Frequencies ◽  
E Coli ◽  
Mutant Strains ◽  
Missense Mutant ◽  
Type Sequence ◽  
Lines Of Evidence

ABSTRACT Recombination frequencies have been determined in crosses involving 28 mutant strains for 20 of which the site of the alteration is known from studies of amino-acid substitutions in the protein products. Three of these mutants showed especially high frequencies of recombination when crossed to other single mutants or when crossed to a strain carrying two alterations at opposite ends of the trpA gene. There is no obvious molecular explanation of the high recombination of these three mutants. They include one missense mutant, one amber and one ochre. The low-frequency recombination mutants include all these same classes as well as frameshift mutants. There is nothing unique about the intragenic location of the high-recombination mutants; in each case there is at least one low-recombination mutant in the same codon.—Crosses involving mutants which were isolated in an altered wild type have shown that the behavior of a high-recombination mutant does not result from its molecular configuration alone, but from its combination with the homologous wild-type sequence from the other parent.—Several lines of evidence indicate that recombination in this system frequently involves closely-spaced double exchanges (about 40 codons apart).

Recombinant Variants of Tissue-Type Plasminogen Activator Containing Amino Acid Substitutions in the Finger Domain

1992 ◽  
Vol 68 (06) ◽  
pp. 672-677 ◽  
Author(s):  
Hitoshi Yahara ◽  
Keiji Matsumoto ◽  
Hiroyuki Maruyama ◽  
Tetsuya Nagaoka ◽  
Yasuhiro Ikenaka ◽  
...  
Keyword(s):  
Amino Acid ◽  
Plasminogen Activator ◽  
Half Life ◽  
Tissue Type ◽  
Clot Lysis ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Plasma Clot ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator

SummaryTissue-type plasminogen activator (t-PA) is a fibrin-specific agent which has been used to treat acute myocardial infarction. In an attempt to clarify the determinants for its rapid clearance in vivo and high affinity for fibrin clots, we produced five variants containing amino acid substitutions in the finger domain, at amino acid residues 7–9, 10–14, 15–19, 28–33, and 37–42. All the variants had a prolonged half-life and a decreased affinity for fibrin of various degrees. The 37–42 variant demonstrated about a 6-fold longer half-life with a lower affinity for fibrin. Human plasma clot lysis assay estimated the fibrinolytic activity of the 37–42 variant to be 1.4-fold less effective than that of the wild-type rt-PA. In a rabbit jugular vein clot lysis model, doses of 1.0 and 0.15 mg/kg were required for about 70% lysis in the wild-type and 37–42 variant, respectively. Fibrinogen was degraded only when the wild-type rt-PA was administered at a dose of 1.0 mg/kg. These findings suggest that the 37–42 variant can be employed at a lower dosage and that it is a more fibrin-specific thrombolytic agent than the wild-type rt-PA.

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