The zinc finger motif of Escherichia coli RecQ is implicated in both DNA binding and protein folding

The RecQ family of DNA helicases has been shown to be important for the maintenance of genomic integrity. Mutations in human RecQ genes lead to genomic instability and cancer. Several RecQ family of helicases contain a putative zinc finger motif of the C4 type at the C terminus that has been identified in the crystalline structure of RecQ helicase from Escherichia coli. To better understand the role of this motif in helicase from E. coli, we constructed a series of single mutations altering the conserved cysteines as well as other highly conserved residues. All of the resulting mutant proteins exhibited a high level of susceptibility to degradation, making functional analysis impossible. In contrast, a double mutant protein in which both cysteine residues Cys397 and Cys400 in the zinc finger motif were replaced by asparagine residues was purified to homogeneity. Slight local conformational changes were detected, but the rest of the mutant protein has a well defined tertiary structure. Furthermore, the mutant enzyme displayed ATP binding affinity similar to the wild-type enzyme but was severely impaired in DNA binding and in subsequent ATPase and helicase activities. These results revealed that the zinc finger binding motif is involved in maintaining the integrity of the whole protein as well as DNA binding. We also showed that the zinc atom is not essential to enzymatic activity.     

Solution structure of the RNA binding domain in the human muscleblind‐like protein 2

The muscleblind‐like (MBNL) proteins 1, 2, and 3, which contain four CCCH zinc finger motifs (ZF1–4), are involved in the differentiation of muscle inclusion by controlling the splicing patterns of several pre‐mRNAs. Especially, MBNL1 plays a crucial role in myotonic dystrophy. The CCCH zinc finger is a sequence motif found in many RNA binding proteins and is suggested to play an important role in the recognition of RNA molecules. Here, we solved the solution structures of both tandem zinc finger (TZF) motifs, TZF12 (comprising ZF1 and ZF2) and TZF34 (ZF3 and ZF4), in MBNL2 from Homo sapiens. In TZF12 of MBNL2, ZF1 and ZF2 adopt a similar fold, as reported previously for the CCCH‐type zinc fingers in the TIS11d protein. The linker between ZF1 and ZF2 in MBNL2 forms an antiparallel β‐sheet with the N‐terminal extension of ZF1. Furthermore, ZF1 and ZF2 in MBNL2 interact with each other through hydrophobic interactions. Consequently, TZF12 forms a single, compact global fold, where ZF1 and ZF2 are approximately symmetrical about the C2 axis. The structure of the second tandem zinc finger (TZF34) in MBNL2 is similar to that of TZF12. This novel three‐dimensional structure of the TZF domains in MBNL2 provides a basis for functional studies of the CCCH‐type zinc finger motifs in the MBNL protein family.     

The primary structure of chicken ribosomal protein L37a.

The amino acid sequence of chicken ribosomal protein L37a was deduced from the nucleotide sequence of a recombinant cDNA and its genomic DNA. Chicken ribosomal protein L37a has 92 amino acids and a molecular mass of 10,247 Da including the initiator methionine. The protein contains a typical Cys2Cys2 zinc finger motif, which may be involved in protein-RNA interaction.     

The Arabidopsis thaliana tandem zinc finger 1 (AtTZF1) protein in RNA binding and decay

The Arabidopsis thaliana tandem zinc finger 1 (AtTZF1) protein is characterized by two tandem‐arrayed CCCH‐type zinc fingers. We have previously found that AtTZF1 affects hormone‐mediated growth, stress and gene expression responses. While much has been learned at the genetic and physiological level, the molecular mechanisms underlying the effects of AtTZF1 on gene expression remain obscure. A human TZF protein, hTTP, is known to bind and trigger the degradation of mRNAs containing AU‐rich elements (AREs) at the 3′ untranslated regions. However, while the TZF motif of hTTP is characterized by C_X8C_X5C_X3H‐_X18‐C_X8C_X5C_X3H, AtTZF1 contains an atypical motif of C_X7C_X5C_X3H‐_X16‐C_X5C_X4C_X3H. Moreover, the TZF motif of AtTZF1 is preceded by an arginine‐rich (RR) region that is unique to plants. Using fluorescence anisotropy and electrophoretic mobility shift binding assays, we have demonstrated that AtTZF1 binds to RNA molecules with specificity and the interaction is dependent on the presence of zinc. Compared with hTTP, in which TZF is solely responsible for RNA binding, both TZF and RR regions of AtTZF1 are required to achieve high‐affinity RNA binding. Moreover, zinc finger integrity is vital for RNA binding. Using a plant protoplast transient expression analysis we have further revealed that AtTZF1 can trigger the decay of ARE‐containing mRNAs in vivo. Taken together, our results support the notion that AtTZF1 is involved in RNA turnover.     

Identification of ZFR, an ancient and highly conserved murine chromosome-associated zinc finger protein

In a screen for RNA binding proteins expressed during murine spermatogenesis, we cloned a novel, ancient zinc finger protein possessing a region common to a small class of RNA binding proteins. Zfr (zinc finger RNA binding) encodes a protein of 1052 amino acids with three widely spaced Cys2His2 zinc fingers. Outside of the zinc fingers, ZFR shares a region that is highly conserved between several RNA binding proteins containing copies of the double-stranded RNA binding motif. By northern blotting, Zfr is expressed at highest levels within the testis, ovary and brain. Immunohistochemistry and confocal microscopy were used to show that ZFR is highly expressed during meiosis I in males and females and is chromosome associated. Zfr is also expressed in Sertoli cells in the testis and granulosa cells in the ovary where it is localized to the nucleus. Using fluorescent in situ hybridization we mapped Zfr to chromosome 15 region A. ZFR appears to be an ancient protein, as apparent homologs exist in invertebrates (D. melanogaster) nematodes (C. elegans) and humans (H. sapiens).     

大肠杆菌YacG锌指结构的金属结合及功能特性

YacG蛋白是一种能够抑制大肠杆菌促旋酶（E.coli gyrase）活性的内源性小分子蛋白质,仅由65 个氨基酸残基组成。核磁共振(NMR)研究发现,YacG结构中含有1个Cys-X2-Cys-X15-Cys-X3-Cys序列的锌指结构域,然而其作用并不清楚。本研究发现,在添加外源锌或者铁的M9基础培养基中,表达并纯化得到分别含有锌和铁的YacG蛋白,而在同时添加铁和L-半胱氨酸的M9基础培养基中可以纯化得到含有铁硫簇的蛋白质。这表明,YacG不仅是一个锌指蛋白,也是铁结合或铁硫簇结合蛋白。定点突变实验发现,YacG锌指结构中的4个半胱氨酸残基突变后,其结合的锌、铁、铁硫簇的含量都显著下降。这提示,锌结合、铁结合以及铁硫簇结合的位点均位于锌指结构域中的4个半胱氨酸残基。体内YacG过表达实验显示,用IPTG在大肠杆菌体内诱导表达野生型YacG蛋白会导致其生长明显受到抑制,而过表达突变体蛋白（YacG-C12/28S）对其生长的抑制作用将会减弱。体外实验进一步发现,锌结合、铁结合以及铁硫簇结合形式的YacG蛋白对E.coli gyrase促DNA螺旋活性的抑制作用没有明显差别,但是锌指结构突变体蛋白（YacG-C12/28S）对gyrase活性的抑制作用显著减弱。这说明,完整的锌指结构对YacG抑制gyrase活性的功能具有重要作用。此研究有可能为gyrase抑制剂类抗生素药物的研发提供有用的线索。     

烟草C2H2锌指蛋白转录因子家族成员的鉴定与表达分析

C2H2锌指蛋白转录因子家族在真核生物中具有重要的生物学功能,广泛参与植物叶的发生、花器官的调控、侧枝的形成及逆境胁迫等生命过程。植物C2H2锌指蛋白不仅结合DNA和RNA,而且与蛋白质之间相互作用。本研究利用普通烟草(Nicotiana tabacum)基因组数据库,运用Blastp比对,结合Pfam和SMART分析,鉴定了118条普通烟草C2H2锌指蛋白家族成员;对烟草C2H2锌指蛋白家族进行了进化树分析、结构域分析、物理化学性质分析、染色体定位、基因结构分析、三维结构分析及组织表达分析等。结果表明：不同成员的氨基酸长度差异较大;系统进化及结构域分析显示,所有C2H2家族成员可以被分为5个亚家族,同一亚家族成员之间在结构域和理化性质上呈现较高一致性;每个成员都含有C2H2结构域,在数量上存在较大差异;将所有基因家族成员定位在22条染色体上;组织表达分析表明,每个C2H2亚家族都有成员在不同组织中表达,在叶及根中有些基因的表达量较高。     

Cloning and characterization of a gene encoding novel zinc finger protein transcription factor induced under water deficit stress from rice (Oryza sativa) cv. N-22

A gene OsZnI encoding Cys3/His1-type zinc finger protein was isolated from the water stress-induced cDNA library of rice (Oryza sativa) cv. N-22, an early maturing, deep-rooted, drought-tolerant genotype adapted to upland conditions. The in-silico analysis revealed an insert of 800 bp with an ORF of 663 nucleotides, encoding 221 amino acids. OsZnI had three distinct features--nuclear localization signal (NLS) present in Arg152-Arg168, Zn finger domain between 185-193 amino acids and 12 amino acids conserved domain in 71-82 amino acids homologous to LEA motif, and belonged to C-type family of Zn finger protein. OsZnI showed induced expression under water deficit stress.     

Solution structures and characterization of human immunodeficiency virus Rev responsive element IIB RNA targeting zinc finger proteins

The Rev responsive element (RRE), a part of unspliced human immunodeficiency virus (HIV) RNA, serves a crucial role in the production of infectious HIV virions. The viral protein Rev binds to RRE and facilitates transport of mRNA to the cytoplasm. Inhibition of the Rev-RRE interaction disrupts the viral life cycle. Using a phage display protocol, dual zinc finger proteins (ZNFs) were generated that bind specifically to RREIIB at the high affinity Rev binding site. These proteins were further shortened and simplified, and they still retained their RNA binding affinity. The solution structures of ZNF29 and a mutant, ZNF29G29R, have been determined by nuclear magnetic resonance (NMR) spectroscopy. Both proteins form C(2)H(2)-type zinc fingers with essentially identical structures. RNA protein interactions were evaluated quantitatively by isothermal titration calorimetry, which revealed dissociation constants (K(d)'s) in the nanomolar range. The interaction with the RNA is dependent upon the zinc finger structure; in the presence of EDTA, RNA binding is abolished. For both proteins, RNA binding is mediated by the alpha-helical portion of the zinc fingers and target the bulge region of RREIIB-TR. However, ZNF29G29R exhibits significantly stronger binding to the RNA target than ZNF29; this illustrates that the binding of the zinc finger scaffold is amenable to further improvements.     

Novel RNA recognition motif domain in the cytoplasmic polyadenylation element binding protein 3

The family of cytoplasmic polyadenylation element binding proteins CPEB1, CPEB2, CPEB3, and CPEB4 binds to the 3′‐untranslated region (3′‐UTR) of mRNA, and plays significant roles in mRNA metabolism and translation regulation. They have a common domain organization, involving two consecutive RNA recognition motif (RRM) domains followed by a zinc finger domain in the C‐terminal region. We solved the solution structure of the first RRM domain (RRM1) of human CPEB3, which revealed that CPEB3 RRM1 exhibits structural features distinct from those of the canonical RRM domain. Our structural data provide important information about the RNA binding ability of CPEB3 RRM1. Proteins 2014; 82:2879–2886. © 2014 Wiley Periodicals, Inc.