共查询到20条相似文献,搜索用时 8 毫秒
1.
RNA recognition motifs (RRMs) constitute versatile macromolecular interaction platforms. They are found in many components of spliceosomes, in which they mediate RNA and protein interactions by diverse molecular strategies. The human U11/U12-65K protein of the minor spliceosome employs a C-terminal RRM to bind hairpin III of the U12 small nuclear RNA (snRNA). This interaction comprises one side of a molecular bridge between the U11 and U12 small nuclear ribonucleoprotein particles (snRNPs) and is reminiscent of the binding of the N-terminal RRMs in the major spliceosomal U1A and U2B″ proteins to hairpins in their cognate snRNAs. Here we show by mutagenesis and electrophoretic mobility shift assays that the β-sheet surface and a neighboring loop of 65K C-terminal RRM are involved in RNA binding, as previously seen in canonical RRMs like the N-terminal RRMs of the U1A and U2B″ proteins. However, unlike U1A and U2B″, some 30 residues N-terminal of the 65K C-terminal RRM core are additionally required for stable U12 snRNA binding. The crystal structure of the expanded 65K C-terminal RRM revealed that the N-terminal tail adopts an α-helical conformation and wraps around the protein toward the face opposite the RNA-binding platform. Point mutations in this part of the protein had only minor effects on RNA affinity. Removal of the N-terminal extension significantly decreased the thermal stability of the 65K C-terminal RRM. These results demonstrate that the 65K C-terminal RRM is augmented by an N-terminal element that confers stability to the domain, and thereby facilitates stable RNA binding. 相似文献
2.
In a continuing effort to explore structural and functional dynamics in RNA catalysis, we have created a series of allosteric hammerhead ribozymes that are activated by theophylline. Representative ribozymes exhibit greater than 3000-fold activation upon effector-binding and cleave with maximum rate constants that are equivalent to the unmodified hammerhead ribozyme. In addition, we have evolved a variant allosteric ribozyme that exhibits an effector specificity change from theophylline to 3-methylxanthine. Molecular discrimination between the two effectors appears to be mediated by subtle conformational differences that originate from displacement of the phosphodiester backbone near the effector binding pocket. These findings reveal the importance of abstruse aspects of molecular recognition by nucleic acids that are likely to be unapproachable by current methods of rational design. 相似文献
3.
Two classes of RNA aptamers that bind the second messenger adenosine 3',5'-cyclic monophosphate (cAMP; 1) were isolated from a random-sequence pool using in vitro selection. Class I and class II aptamers are formed by 33- and 31-nucleotide RNAs, respectively, and each is comprised of similar stem-loop and single-stranded structural elements. Class II aptamers, which dominate the final selected RNA population, require divalent cations for complex formation and display a dissociation constant (K(D)) for cAMP of approximately 10 microM. A representative class II aptamer exhibits substantial discrimination against 5'- and 3'-phosphorylated nucleosides such as ATP, 5'-AMP, and 3'-AMP. However, components of cAMP such as adenine and adenosine also are bound, indicating that the adenine moiety is the primary positive determinant of ligand binding. Specificity of cAMP binding appears to be established by hydrogen bonding interactions with the adenine base as well as by steric interactions with groups on the ribose moiety. In addition, the aptamer recognizes 8,5'-O-cycloadenosine (2) but not N(3), 5'-cycloadenosine (3), indicating that this RNA might selectively recognize the anti conformation of the N-glycosidic bond of cAMP. 相似文献
4.
Co(2+) binding RNA aptamers were chosen as research models to reveal the structural basis underlying the recognition of Co(2+) by RNA, with the application of two distinct methods. Using the nucleotide analog interference mapping assay, we found strong interference effects after incorporation of the 7-deaza guanosine phosphorotioate analog into the RNA chain at equivalent positions G27 and G28 in aptamer no. 18 and G25 and G26 in aptamer no. 20. The results obtained by nucleotide analog interference mapping suggest that these guanine bases are crucial for the creation of Co(2+) binding sites and that they appear to be involved in the coordination of the ion to the exposed N7 atom of the tandem guanines. Additionally, most 7-deaza guanosine phosphorotioate and 7-deaza adenosine phosphorotioate interferences were located in the common motifs: loop E-like in aptamer no. 18 and kissing dimer in aptamer no. 20. We also found that purine-rich stretches containing guanines with the highest interference values were the targets for hybridization of 6-mers, which are members of the semi-random oligodeoxyribonucleotide library in both aptamers. It transpired that DNA oligomer directed RNase H digestions are sensitive to Co(2+) and, at an elevated metal ion concentration, the hybridization of oligomers to aptamer targets is inhibited, probably due to higher stability and complexity of the RNA structure. 相似文献
5.
Cerdà-Costa N Bonet J Fernández MR Avilés FX Oliva B Villegas S 《Journal of molecular modeling》2011,17(8):1863-1875
The observation that activation domains (AD) of procarboxypeptidases are rather long compared to the pro-regions of other
zymogens raises the possibility that they could play additional roles apart from precluding enzymatic activity within the
proenzyme and helping in its folding process. In the present work, we compared the overall pro-domain tertiary structure with
several proteins belonging to the same fold in the structural classification of proteins (SCOP) database by using structure
and sequence comparisons. The best score obtained was between the activation domain of human procarboxypeptidase A4 (ADA4h)
and the human U1A protein from the U1 snRNP. Structural alignment revealed the existence of RNP1- and RNP2-related sequences
in ADA4h. After modeling ADA4h on U1A, the new structure was used to extract a new sequence pattern characteristic for important
residues at key positions. The new sequence pattern allowed scanning protein sequences to predict the RNA-binding function
for 32 sequences undetected by PFAM. Unspecific RNA electrophoretic mobility shift assays experimentally supported the prediction
that ADA4h binds an RNA motif similar to the U1A binding-motif of stem-loop II of U1 small nuclear RNA. The experiments carried
out with ADA4h in the present work suggest the sharing of a common ancestor with other RNA recognition motifs. However, the
fact that key residues preventing activity within the proenzyme are also key residues for RNA binding might have induced the
activation domains of procarboxypeptidases to evolve from the canonical RNP1 and RNP2 sequences. 相似文献
6.
A randomly generated mutation in Escherichia coli alanine tRNA synthetase compensates for a mutation in its cognate tRNA. The enzyme's mutation occurs next to a Cys-X2-Cys-X6-His-X2-His metal-binding motif that is distinct from the zinc finger motif found in some DNA-binding proteins. Instead, the synthetase's metal binding domain resembles the Cys-X2-Cys-X4-His-X4-Cys metal-binding domain of the gag gene product of retroviruses. For Ala-tRNA synthetase, the metal bound at the Cys-His motif is important specifically for the tRNA-dependent step of catalysis, and the enzyme-tRNA interaction is dependent on the geometry of metal co-ordination to the enzyme. These data, and the demonstrated sensitivity of RNA packaging to mutations in the metal-binding domain of the gag gene product of retroviruses, suggest that an aminoacyl-tRNA synthetase and retroviruses have adopted a related metal-binding motif for RNA recognition. 相似文献
7.
In yeast (Saccharomyces cerevisiae), the branchpoint binding protein (BBP) recognizes the conserved yeast branchpoint sequence (UACUAAC) with a high level of specificity and affinity, while the human branchpoint binding protein (SF1) binds the less-conserved consensus branchpoint sequence (CURAY) in human introns with a lower level of specificity and affinity. To determine which amino acids in BBP provide the additional specificity and affinity absent in SF1, a panel of chimeric SF1 proteins was tested in RNA binding assays with wild-type and mutant RNA substrates. This approach revealed that the QUA2 domain of BBP is responsible for the enhanced RNA binding affinity and specificity displayed by BBP compared with SF1. Within the QUA2 domain, a transposition of adjacent arginine and lysine residues is primarily responsible for the switch in RNA binding between BBP and SF1. Alignment of multiple branchpoint binding proteins and the related STAR/GSG proteins suggests that the identity of these two amino acids and the RNA target sequences of all of these proteins are correlated. 相似文献
8.
9.
The RNA recognition motif (RRM) is one of the most common eukaryotic protein motifs. RRM sequences form a conserved globular structure known as the RNA-binding domain (RBD) or the ribonucleoprotein domain. Many proteins that contain RRM sequences bind RNA in a sequence-specific manner. To investigate the basis for the RNA-binding specificity of RRMs, we subjected 330 aligned RRM sequences to covariance analysis. The analysis revealed a single network of covariant amino acid pairs comprising the buried core of the RBD and a surface patch. Structural studies have implicated a subset of these residues in RNA binding. The covariance linkages identify a larger set of amino acid residues, including some not directly in contact with bound RNA, that may influence RNA-binding specificity. 相似文献
10.
11.
12.
A novel neomycin–methidium conjugate was synthesized. The covalent linkage of the aminoglycoside to an intercalator, a derivative of ethidium bromide, results in a new conjugate capable of selective recognition of the DNA:RNA hybrid duplex. Spectroscopic methods: UV, CD, fluorescence, and calorimetric techniques: DSC and ITC were used to characterize the sub-nanomolar binding displayed by the conjugate for the DNA:RNA hybrid duplex, poly(dA):poly(rU). 相似文献
13.
Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is a multitasking protein involved in RNA packaging, alternative splicing of pre-mRNA, telomere maintenance, cytoplasmic RNA trafficking, and translation. It binds short segments of single-stranded nucleic acids, including the A2RE11 RNA element that is necessary and sufficient for cytoplasmic transport of a subset of mRNAs in oligodendrocytes and neurons. We have explored the structures of hnRNP A2, its RNA recognition motifs (RRMs) and Gly-rich module, and the RRM complexes with A2RE11. Circular dichroism spectroscopy showed that the secondary structure of the first 189 residues of hnRNP A2 parallels that of the tandem betaalpha betabeta alphabeta RRMs of its paralogue, hnRNP A1, previously deduced from X-ray diffraction studies. The unusual GRD was shown to have substantial beta-sheet and beta-turn structure. Sedimentation equilibrium and circular dichroism results were consistent with the tandem RRM region being monomeric and supported earlier evidence for the binding of two A2RE11 oligoribonucleotides to this domain, in contrast to the protein dimer formed by the complex of hnRNP A1 with the telomeric ssDNA repeat. A three-dimensional structure for the N-terminal, two-RRM-containing segment of hnRNP A2 was derived by homology modeling. This structure was used to derive a model for the complex with A2RE11 using the previously described interaction of pairs of stacked nucleotides with aromatic residues on the RRM beta-sheet platforms, conserved in other RRM-RNA complexes, together with biochemical data and molecular dynamics-based observations of inter-RRM mobility. 相似文献
14.
Jin H Sepúlveda J Burrone OR 《Protein science : a publication of the Protein Society》2004,13(12):3222-3229
Anti-DNA antibodies have the potential to be applied in vast fields of fundamental as well as medical research. They are found in autoimmune diseases, such as systemic lupus erythemotosus. In most cases, anti-dsDNA antibodies do not present sequence specificity and are of low affinity. The dominant role of VH domains in DNA recognition induced us to search for binders based on VH dimers (VHD), previously reported to bind different protein antigens. We screened a phage displayed homo-VHD library against a 19-bp dsDNA sequence. A sequence-specific binder was selected, which recognizes the terminal located CTGC motif with a Kd of 250 nM. Association of the two identical VH domains of the molecule was shown to be essential for binding. 相似文献
15.
Grouping of amino acids and recognition of protein structurally conserved regions by reduced alphabets of amino acids
下载免费PDF全文
![点击此处可从《中国科学C辑(英文版)》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Sequence alignment is a common method for finding protein structurally conserved/similar regions. However, sequence alignment
is often not accurate if sequence identities between to-be-aligned sequences are less than 30%. This is because that for these
sequences, different residues may play similar structural roles and they are incorrectly aligned during the sequence alignment
using substitution matrix consisting of 20 types of residues. Based on the similarity of physicochemical features, residues
can be clustered into a few groups. Using such simplified alphabets, the complexity of protein sequences is reduced and at
the same time the key information encoded in the sequences remains. As a result, the accuracy of sequence alignment might
be improved if the residues are properly clustered. Here, by using a database of aligned protein structures (DAPS), a new
clustering method based on the substitution scores is proposed for the grouping of residues, and substitution matrices of
residues at different levels of simplification are constructed. The validity of the reduced alphabets is confirmed by relative
entropy analysis. The reduced alphabets are applied to recognition of protein structurally conserved/similar regions by sequence
alignment. The results indicate that the accuracy or efficiency of sequence alignment can be improved with the optimal reduced
alphabet with N around 9. 相似文献
16.
Sequence alignment is a common method for finding protein structurally conserved/similar regions. However, sequence alignment
is often not accurate if sequence identities between to-be-aligned sequences are less than 30%. This is because that for these
sequences, different residues may play similar structural roles and they are incorrectly aligned during the sequence alignment
using substitution matrix consisting of 20 types of residues. Based on the similarity of physicochemical features, residues
can be clustered into a few groups. Using such simplified alphabets, the complexity of protein sequences is reduced and at
the same time the key information encoded in the sequences remains. As a result, the accuracy of sequence alignment might
be improved if the residues are properly clustered. Here, by using a database of aligned protein structures (DAPS), a new
clustering method based on the substitution scores is proposed for the grouping of residues, and substitution matrices of
residues at different levels of simplification are constructed. The validity of the reduced alphabets is confirmed by relative
entropy analysis. The reduced alphabets are applied to recognition of protein structurally conserved/similar regions by sequence
alignment. The results indicate that the accuracy or efficiency of sequence alignment can be improved with the optimal reduced
alphabet with N around 9.
Supported by the National Natural Science Foundation of China (Grant Nos. 90403120, 10474041 and 10021001) and the Nonlinear
Project (973) of the NSM 相似文献
17.
18.
19.