Privileged chiral catalysts

One of the most active current areas of chemical research is centered on how to synthesize handed (chiral) compounds in a selective manner, rather than as mixtures of mirror-image forms (enantiomers) with different three-dimensional structures (stereochemistries). Nature points the way in this endeavor: different enantiomers of a given biomolecule can exhibit dramatically different biological activities, and enzymes have therefore evolved to catalyze reactions with exquisite selectivity for the formation of one enantiomeric form over the other. Drawing inspiration from these natural catalysts, chemists have developed a variety of synthetic small-molecule catalysts that can achieve levels of selectivity approaching, and in some cases matching, those observed in enzymatic reactions.     

Metals and DNA: molecular left-handed complements

Chiral metal complexes provide unique molecular probes for DNA. Chiral reagents that "recognize" different local structures along the DNA strand have been designed by a process in which the asymmetry in shape and size of the complex is matched to that of the DNA helical groove. As a result, the chiral metal complexes provide very sensitive probes for local helical structure, both left- and right-handed. Direct coordination of chiral complexes to the DNA bases adds an element of sequence selectivity to the probe design. With a suitable reactive metal center, reagents that target chemically specific sites along the strand may be developed. One such chiral reagent, which cleaves left-handed DNA sites with photoactivation, has been useful in mapping this distinct conformation and examining its biological role. The conformation-specific molecular cleaver, much like a DNA-binding enzyme, recognizes and reacts at discrete sites along the DNA strand. These site-specific chiral metal complexes provide exciting new tools for probing the local variations in DNA structure and its role in the regulation of gene expression.     

Cloning of a membrane protein that induces a slow voltage-gated potassium current

A rat kidney messenger RNA that induces a slowly activating, voltage-dependent potassium current on its expression in Xenopus oocytes was identified by combining molecular cloning with an electrophysiological assay. The cloned complementary DNA encodes a novel membrane protein that consists of 130 amino acids with a single putative transmembrane domain. This protein differs from the known ion channel proteins but is involved in the induction of selective permeation of potassium ions by membrane depolarization.     

Regulation of bacterial growth

Is the control of bacterial metabolism so complex? The answer can be found in a simple experiment. Two cultures of bacteria are grown in different mediums. One contains as the carbon and nitrogen sources a mixture of amino acids, while the other contains only glucose and ammonia, so that the cells must synthesize all of the amino acids. The results show that insofar as the cells in both cultures grow at comparable rates, they will have the same composition in terms of DNA, RNA, and protein (30). To explain this phenomena I have argued that through the control mechanisms responsible for the distribution of substrates in intermediary metabolism, the substrates of protein synthesis are produced at concentrations and rates commensurate with the ability of the environment to support growth. The provision of these substrates relative to the ability of the protein forming system to utilize them regulates the synthesis of ribosomal and transfer RNA, which, after adjustment for various modulating influences, such as nonfunctioning ribosomes or ribosomal RNA turnover, brings the number of functioning ribosomes to a point in keeping with the provision of external nutrients. The synthesis of messenger (or total) RNA, ribosomal proteins, and DNA, and the process of cell division, for example, are subject to their own controls, but through the burden they each place on intermediary metabolism, they provide a means for partitioning the cell's metabolic resources. It might be noted that this view may not be very far from the idea once held that the rate at which each of the transfer RNA's was changed by amino acids regulate the synthesis of bacterial RNA, but growth regulation is clearly more complicated than implied by that model (76).     

Polypeptide sequences essential for RNA recognition by an enzyme

Many RNAs are complex, globular molecules formed from elements of secondary and tertiary structure analogous to those found in proteins. Little is known about recognition of RNAs by proteins. In the case of transfer RNAs (tRNAs), considerable evidence suggests that elements dispersed in both the one- and three-dimensional structure are important for recognition by aminoacyl tRNA synthetases. Fragments of alanine tRNA synthetase were created by in vitro manipulations of the cloned alaS gene and examined for their interaction with alanine-specific tRNA. Sequences essential for recognition were located near the middle of the polypeptide, juxtaposed to the carboxyl-terminal side of the domain for aminoacyl adenylate synthesis. The most essential part of the tRNA interaction strength and specificity was dependent on a sequence of fewer than 100 amino acids. Within this sequence, and in the context of the proper conformation, a segment of no more than 17 amino acids was responsible for 25% or more of the total synthetase-tRNA free energy of association. The results raise the possibility that an important part of specific RNA recognition by an aminoacyl tRNA synthetase involves a polypeptide segment that is short relative to the total size of the protein.     

Nucleotide sequence of yellow fever virus: implications for flavivirus gene expression and evolution

The sequence of the entire RNA genome of the type flavivirus, yellow fever virus, has been obtained. Inspection of this sequence reveals a single long open reading frame of 10,233 nucleotides, which could encode a polypeptide of 3411 amino acids. The structural proteins are found within the amino-terminal 780 residues of this polyprotein; the remainder of the open reading frame consists of nonstructural viral polypeptides. This genome organization implies that mature viral proteins are produced by posttranslational cleavage of a polyprotein precursor and has implications for flavivirus RNA replication and for the evolutionary relation of this virus family to other RNA viruses.     

A kinetic partitioning model of selective binding of nonnative proteins by the bacterial chaperone SecB

An in vitro assay for the interaction of SecB, a molecular chaperone from Escherichia coli, with polypeptide ligands was established based on the ability of SecB to block the refolding of denatured maltose-binding protein. Competition experiments show that SecB binds selectively to nonnative proteins with high affinity and without specificity for a particular sequence of amino acids. It is proposed that selectivity in binding is due to a kinetic partitioning of polypeptides between folding and association with SecB.     

Deletion in cysteine-rich region of LDL receptor impedes transport to cell surface in WHHL rabbit

The Watanabe heritable hyperlipidemic (WHHL) rabbit, an animal with familial hypercholesterolemia, produces a mutant receptor for plasma low-density lipoprotein (LDL) that is not transported to the cell surface at a normal rate. Cloning and sequencing of complementary DNA's from normal and WHHL rabbits, shows that this defect arises from an in-frame deletion of 12 nucleotides that eliminates four amino acids from the cysteine-rich ligand binding domain of the LDL receptor. A similar mutation, detected by S1 nuclease mapping of LDL receptor messenger RNA, occurred in a patient with familial hypercholesterolemia whose receptor also fails to be transported to the cell surface. These findings suggest that animal cells may have fail-safe mechanisms that prevent the surface expression of improperly folded proteins with unpaired or improperly bonded cysteine residues.     

Cloning and sequencing of porcine LH-hCG receptor cDNA: variants lacking transmembrane domain

Complementary DNA clones, encoding the LH-hCG (luteinizing hormone-human choriogonadotropic hormone) receptor were isolated by screening a lambda gt11 library with monoclonal antibodies. The primary structure of the protein was deduced from the DNA sequence analysis; the protein contains 696 amino acids with a putative signal peptide of 27 amino acids. Hydropathy analysis suggests the existence of seven transmembrane domains that show homology with the corresponding regions of other G protein-coupled receptors. Three other types of clones corresponding to shorter proteins were observed, in which the putative transmembrane domain was absent. These probably arose through alternative splicing. RNA blot analysis showed similar patterns in testis and ovary with a major RNA of 4700 nucleotides and several minor species. The messenger RNA was expressed in COS-7 cells, yielding a protein that bound hCG with the same affinity as the testicular receptor.     

Expression,regulation and binding affinity of fatty acid-binding protein 2 in Spodoptera litura

Fatty acid-binding proteins(FABPs) are a family of lipid chaperones, which contribute to systemic metabolic regulation through diverse lipid signalings. In this study, a midgut-specific FABP gene(Slfabp2) was cloned from Spodoptera litura. RT-PCR and Western blot analysis indicated that RNA and protein levels of SlFABP2 gradually increased and reached a peak at the prepupal stage and maintained a high level during the pupal stage. The expression of SlFABP2 protein was induced by starvation treatment. In vitro binding assay revealed that the recombinant SlFABP2 had high affinities of binding long-chain fatty acids, such as palmitic acid, arachidonate and oleic acid. The results suggest that SlFABP2 may have a unique function that transports intracellular fatty acids and can regulate the metabolism of lipids in metamorphosis. This work provides experimental clues for understanding the potential function of SlFABP2 in fatty acid metabolism in S. litura.     

Molecular biology. Creation's seventh day

Scientists at the Scripps Research Institute are attempting to find out what life would look like if DNA contained more than four nucleotide bases and proteins more than 20 amino acids. By reengineering DNA, RNA, and the proteins that interact with them, they hope to create synthetic organisms with a chemical makeup fundamentally different from all life that has existed on Earth for the last 3.8 billion years. If they succeed, their biochemical reengineering could have a profound effect on everything from basic molecular biology to industrial chemistry.     

A continuous cell-free translation system capable of producing polypeptides in high yield

A cell-free translation system has been constructed that uses a continuous flow of the feeding buffer [including amino acids, adenosine triphosphate (ATP), and guanosine triphosphate (GTP)] through the reaction mixture and a continuous removal of a polypeptide product. Both prokaryotic (Escherichia coli) and eukaryotic (wheat embryos, Triticum sp.) versions of the system have been tested. In both cases the system has proven active for long times, synthesizing polypeptides at a high constant rate for tens of hours. With the use of MS2 phage RNA or brome mosaic virus RNA 4 as templates, 100 copies of viral coat proteins per RNA were synthesized for 20 hours in the prokaryotic or eukaryotic system, respectively. With synthetic calcitonin messenger RNA, 150 to 300 copies of calcitonin polypeptide were produced per messenger RNA in both types of continuous translation systems for 40 hours.     

Binding of the human Prp31 Nop domain to a composite RNA-protein platform in U4 snRNP

Although highly homologous, the spliceosomal hPrp31 and the nucleolar Nop56 and Nop58 (Nop56/58) proteins recognize different ribonucleoprotein (RNP) particles. hPrp31 interacts with complexes containing the 15.5K protein and U4 or U4atac small nuclear RNA (snRNA), whereas Nop56/58 associate with 15.5K-box C/D small nucleolar RNA complexes. We present structural and biochemical analyses of hPrp31-15.5K-U4 snRNA complexes that show how the conserved Nop domain in hPrp31 maintains high RNP binding selectivity despite relaxed RNA sequence requirements. The Nop domain is a genuine RNP binding module, exhibiting RNA and protein binding surfaces. Yeast two-hybrid analyses suggest a link between retinitis pigmentosa and an aberrant hPrp31-hPrp6 interaction that blocks U4/U6-U5 tri-snRNP formation.     

牙鲆Mx基因的克隆及序列分析

对牙鲆(Paralichthys lethostigma)总RNA经反转录得到的cDNA测序。结果表明,获得的cDNA片段全长1 980 bp,其中编码区长1 890 bp,编码629个氨基酸残基,推测蛋白质分子量大小为71.7 kDa。Blast比较以及Megalign软件分析的结果表明,此cDNA片段具有脊椎动物Mx蛋白共有的结构特征:一个三联体GTP结合区域;一个发动蛋白家族的典型结构特征序列及C端高度保守的Leu拉链结构区。进一步的序列比较与进化分析结果表明,牙鲆Mx基因与日本比目鱼等鱼类的同源性较高,同源性达75.1%-98.7%;而与哺乳动物的同源性相对较低,同源性达50.3%-55.1%。     

RNA mimics of green fluorescent protein

Green fluorescent protein (GFP) and its derivatives have transformed the use and analysis of proteins for diverse applications. Like proteins, RNA has complex roles in cellular function and is increasingly used for various applications, but a comparable approach for fluorescently tagging RNA is lacking. Here, we describe the generation of RNA aptamers that bind fluorophores resembling the fluorophore in GFP. These RNA-fluorophore complexes create a palette that spans the visible spectrum. An RNA-fluorophore complex, termed Spinach, resembles enhanced GFP and emits a green fluorescence comparable in brightness with fluorescent proteins. Spinach is markedly resistant to photobleaching, and Spinach fusion RNAs can be imaged in living cells. These RNA mimics of GFP provide an approach for genetic encoding of fluorescent RNAs.     

Active disruption of an RNA-protein interaction by a DExH/D RNA helicase

All aspects of cellular RNA metabolism and the replication of many viruses require DExH/D proteins that manipulate RNA in a manner that requires nucleoside triphosphates. Although DExH/D proteins have been shown to unwind purified RNA duplexes, most RNA molecules in the cellular environment are complexed with proteins. It has therefore been speculated that DExH/D proteins may also affect RNA-protein interactions. We demonstrate that the DExH protein NPH-II from vaccinia virus can displace the protein U1A from RNA in an active adenosine triphosphate-dependent fashion. NPH-II increases the rate of U1A dissociation by more than three orders of magnitude while retaining helicase processivity. This indicates that DExH/D proteins can effectively catalyze protein displacement from RNA and thereby participate in the structural reorganization of ribonucleoprotein assemblies.     

油松种子萌发过程中核酸和蛋白质的变化

随油松种子萌发的进程,种子内核酸总量、DNA,RNA和蛋白质含量发生不同程度的消长,胚蛋白组分的变化尤为明显,其中低等电点(pH4.3～5.2)区域的酸性蛋白质不断增加和累积;与此相反,高等电点(pH6.5～8.0)低分子量(4.3×10~4D以下)区域的碱性蛋白质逐渐降解、消失,用PAS反应证明它们是一组分子量各异的糖蛋白。种胚内的这些在不同时期出现和消失的蛋白质都具有萌发时期的特征性,它们可能对调节胚的生长和幼苗的形态建成有重要作用。     

金鱼HSC70和HSP40基因克隆及其原核表达

[目的]克隆金鱼热激同源蛋白70(HSC70)和热休克蛋白(HSP40)基因,构建原核表达载体并通过IPTG诱导表达获得目的蛋白,明确HSC70和HSP40蛋白的生物学功能,为揭示金鱼的高温应答机制打下基础.[方法]提取金鱼鳃组织总RNA,利用RT-PCR扩增金鱼HSC70和HSP40基因,将目的基因片段克隆至线性空表达载体pET-32a上构建原核表达载体pET-32a-HSC70和pET-32a-HSP40,转化大肠杆菌Rosetta表达菌株后用IPTG进行诱导表达,并以SDS-PAGE和Western blotting检测分析表达获得的融合蛋白.[结果]金鱼HSC70基因CDS序列全长1950 bp,编码650个氨基酸;金鱼HSP40基因CDS序列全长996 bp,编码332个氨基酸.金鱼HSC70氨基酸序列与斑马鱼、人类、家鼠、金线鲃和草鱼的相似度分别为96.28%、95.50%、95.35%、98.15%和98.61%;HSP40氨基酸序列与斑马鱼的完全一致(相似度100.00%),与普通鲤鱼、金线鲃、人类和家鼠的相似度均为94.12%.原核诱导表达获得的融合蛋白Trx-HSC70和Trx-HSP40为可溶性表达,其大小约91.5和55.0 kD,均能与Anti-Trx抗体发生特异性反应.[结论]HSC70和HSP40蛋白在鱼类及哺乳动物中高度保守,经原核诱导表达获得的金鱼融合蛋白Trx-HSC70和Trx-HSP40为可溶性表达,且携带有Trx标签,能与Anti-Trx抗体发生特异性反应,可作为互作蛋白用于RNA pull down试验.     

Molecular biology. Unwinding RNA silencing

Eukaryotic cells have developed an elegant system called RNA silencing for getting rid of foreign RNAs whether they be of viral, retrotransposon, or transgene origin. In his Perspective, Baulcombe examines new findings (Wu-Scharf et al.) showing that in a green alga the gene responsible for RNA silencing encodes an RNA helicase (related to proteins in worms and other organisms) that is required for regulation of gene expression at the RNA level.