The mouse quaking (qk) gene is essential in both myelination and early embryogenesis. Its product, QKI, is an RNA-binding protein belonging to a growing protein family called STAR (signal transduction and activator of RNA). All members have an ϳ200-amino acid STAR domain, which contains a single extended heteronuclear ribonucleoprotein K homologue domain flanked by two domains called QUA1 and QUA2. We found that QKI isoforms could associate with each other, while one of the lethal mutations qkI kt4 with a single amino acid change in QUA1 domain, leads to a loss of QKI selfinteraction. This suggests that the QUA1 domain is responsible for QKI dimerization. Three QKI isoforms have different carboxyl termini and different subcellular localization. Here, using GFP fusion protein, we identified a 7-amino acid novel nuclear localization sequence in the carboxyl terminus of QKI-5, which is conserved in a subclass of STAR proteins containing SAM68 and ETLE/T-STAR. Thus, we name this motif STAR-NLS. In addition, the effects of active transcription, RNAbinding and self-interaction on QKI-5 localization were analyzed. Furthermore, using an interspecies heterokaryon assay, we found that QKI-5, but not another STAR protein ETLE, shuttles between the nucleus and the cytoplasm, which suggests that QKI-5 functions in both cell compartments.

Detailed characterization of post-translational modifications (PTMs) of proteins in microbial communities remains a significant challenge. Here we directly identify and quantify a broad range of PTMs (hydroxylation, methylation, citrullination, acetylation, phosphorylation, methylthiolation, S-nitrosylation and nitration) in a natural microbial community from an acid mine drainage site. Approximately 29% of the identified proteins of the dominant Leptospirillum group II bacteria are modified, and 43% of modified proteins carry multiple PTM types. Most PTM events, except S-nitrosylations, have low fractional occupancy. Notably, PTM events are detected on Cas proteins involved in antiviral defense, an aspect of Cas biochemistry not considered previously. Further, Cas PTM profiles from Leptospirillum group II differ in early versus mature biofilms. PTM patterns are divergent on orthologues of two closely related, but ecologically differentiated, Leptospirillum group II bacteria. Our results highlight the prevalence and dynamics of PTMs of proteins, with potential significance for ecological adaptation and microbial evolution.

We have employed a cell-free human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) assay to study the effects of non-nucleoside inhibitors of RT (NNRTI) by directly monitoring specific HIV DNA products using a HIV-1 genome-derived template and an oligodeoxynucleotide primer. As previously shown by ourselves and others, nucleoside analog triphosphates, e.g. 3-azido-3-deoxythymidine triphosphate and 2,3-dideoxyadenosine triphosphate, could directly inhibit HIV RT RNA-dependent DNA polymerase activity by causing chain termination, as visualized in a RT reaction that yields specific DNA products. In contrast, each of two NNRTIs, nevirapine and delavirdine, directly inhibited RT activity without causing chain termination effects. We also analyzed interactions between nucleoside analogs and NNRTIs or among NNRTIs by chain elongation/dNTP incorporation and/or steady-state kinetic assays. Combinations of nevirapine with the triphosphates of either the (؊)-strand of 2,3-dideoxy-3-thiacytidine or 2,3-dideoxyadenosine yielded additive/ synergistic effects on RT activity. However, only an additive effect was observed when combinations of nevirapine and 3-azido-3-deoxythymidine triphosphate were employed. Combinations of nevirapine and delavirdine had an antagonistic effect on the inhibition of HIV-1 RT activity.A key step in the HIV 1 life cycle is the reverse transcription of genomic RNA into double-stranded DNA mediated by the viral-encoded multifunctional enzyme reverse transcriptase (RT) (1). Two major groups of HIV-1 RT inhibitors have been studied, nucleoside analogs and a series of non-nucleoside RT inhibitors (NNRTI). The former, such as 3Ј-azido-3Ј-deoxythymidine (AZT), 2Ј,3Ј-dideoxyinosine, and the (Ϫ)-strand of 2Ј,3Ј-dideoxy-3Ј-thiacytidine are believed to block HIV-1 replication by competitively inhibiting incorporation of nucleotide substrates by RT and causing chain termination after being incorporated in phosphorylated form into elongating viral DNA (2, 3). NNRTIs are thought to act noncompetitively by binding to a hydrophobic pocket located near the polymerase catalytic site, resulting in inhibition of RT polymerase activity (4 -7). Although these two classes of drugs can generally reduce viral load, at least transiently, and may improve quality of life, problems of drug intolerance and resistance have arisen after long term therapy (8 -10). A series of mutations in viral RT are responsible for resistance to both nucleoside compounds and NNRTIs (11-17), although no cross-resistance has been demonstrated among these two groups of inhibitors (14,18,19).Several studies have shown that recombinant viruses that contained resistance-conferring mutations for each of nucleosides and NNRTIs possessed diminished sensitivity for both groups of compounds (19). Combinations of drugs of both types have yielded synergistic results on the inhibition of HIV-1 replication in tissue culture assays (20 -22). Various combinations of nucleosides and NNRTIs are currently undergoing clinical evaluation.To better un...

Avermectins, a group of polyketide natural products, are widely used as anthelmintics in agriculture. Metabolic engineering and combinatorial biosynthesis were extensively employed to improve Avermectins production and create novel Avermectin derivatives, including Ivermectin and Doramectin. It is labor intensive and time cost to genetically manipulate Avermectins producer Streptomyces avermitilis in vivo. Cloning and heterologous expression of Avermectins biosynthetic gene cluster will make it possible to tailor the cluster in vitro. We constructed a Bacterial Artificial Chromosome (BAC) library of S. avermitilis ATCC 31267 with inserted DNA fragments ranged from 100 to 130 Kb. Five recombinant BAC clones which carried the Avermectins biosynthetic gene cluster ave (81 Kb in size) were screened out from the library. Then, ave was hetero-expressed in S. lividans. Three Avermectin components, A2a, B1a and A1a were detected from the cell extracts of recombinant strains. It will facilitate the development of Avermectin derivatives by polyketide synthase domain swapping and provide functional element for Avermectins synthetic biology study.

Cell 114:623-634, 2003). We show that rraA is expressed from its own promoter, P rraA , located in the menA-rraA intergenic region. Primer extension and lacZ fusion analysis revealed that transcription from P rraA is elevated upon entry into stationary phase in a s -dependent manner. In addition, the stability of the rraA transcript is dependent on RNase E activity, suggesting the involvement of a feedback circuit in the regulation of the RraA level in E. coli.RNase E is an essential protein that plays a crucial role in global mRNA metabolism as well as in the maturation of functional RNAs such as rRNAs, tRNAs, tmRNA, and small regulatory RNAs (3,9,18,19,21,28). To date, RNase E homologs have been found in more than 50 eubacteria, archaebacteria, and plants (16). The cellular level and activity of RNase E are subjected to multiple environmental controls. At one level, RNase E synthesis is autoregulated by modulating the half-life of its own mRNA (12,26). In addition, recent studies have revealed that 5Ј-monophosphorylated RNA serves as an allosteric activator of the endonuclease activity (13). Furthermore, the degradation of target RNAs by RNase E is found to be affected globally by endoribonuclease-binding proteins that control the decay and abundance of individual bacterial mRNAs in trans (8,17).RraA (regulator of ribonuclease activity A), is an evolutionarily conserved 17.4-kDa protein with close homologs (Ͼ40% amino acid identity) in bacteria, archaea, proteobacteria, and plants. RraA binds to RNase E with an equilibrium dissociation constant (K D ) in the low-micromolar range and serves as a trans-acting modulator of the endonuclease activity of the enzyme (17). High-affinity binding requires the C-terminal half region of RNase E, which acts as a scaffold for the assembly of a large multiprotein complex called the degradosome (17,36). RraA appears to interact only with the enzyme and not with RNA substrates (17). Gene chip analysis revealed that the action of RraA results in a dramatic change in the global abundance of mRNAs in Escherichia coli, affecting over 15% of all cellular transcripts. Importantly, the gene expression profile that is obtained upon overexpression of RraA is distinct from that obtained upon depletion of RNase E or through the action of RraB, a second trans-acting RNase E inhibitor of E. coli (8).The rraA gene is located downstream of menA, which encodes a 1,4-dihydroxy-2-naphthoic acid octaprenyltransferase that catalyzes the prenylation of the redox mediator menaquinone (32). Transcription of menA appears to occur from a 70 -dependent promoter. Earlier, Meganathan proposed that rraA (formerly designated menG) is transcribed from the menA promoter in a dicistronic mRNA (22). In this study we demonstrate that rraA is transcribed predominantly from its own promoter (P rraA ) located in the intergenic region between the menA and rraA genes. Transcription from P rraA is s dependent and is induced upon entry into stationary phase. Furthermore, we show that the synthesis of RraA is regulated...

The Escherichia coli RNA degradosome is a protein complex that plays a critical role in the turnover of numerous RNAs. The key component of the degradosome complex is the endoribonuclease RNase E, a multidomain protein composed of an N-terminal catalytic region and a C-terminal region that organizes the other protein components of the degradosome. Previously, the RNase E inhibitors RraA and RraB were identified genetically and shown to bind to the C-terminal region of RNase E, thus affecting both the protein composition of the degradosome and the endonucleolytic activity of RNase E. In the present work, we investigated the transcriptional regulation of rraB. rraB was shown to be transcribed constitutively from its own promoter, PrraB. Transposon mutagenesis and screening for increased ␤-galactosidase activity from a chromosomal PrraB-lacZ transcriptional fusion resulted in the isolation of a transposon insertion in glmS, encoding the essential enzyme glucosamine-6-phosphate synthase that catalyzes the first committed step of the uridine 5-diphospho-N-acetyl-glucosamine (UDP-GlcNAc) pathway, which provides intermediates for peptidoglycan biogenesis. The glmS852::Tn5 allele resulted in an approximately 50% lower intracellular concentration of UDP-GlcNAc and conferred a fivefold increase in the level of rraB mRNA. This allele also mediated a twofold increase in ␤-galactosidase activity from a chromosomal fusion of the 5 untranslated region of the rne gene to lacZ, suggesting that a reduction in cellular concentration of UDP-GlcNAc and the resulting increased expression of RraB might modulate the action of RNase E.The endoribonuclease RNase E plays a central role in RNA metabolism, including the processing of rRNAs and tRNAs (30,42,43); the degradation of small regulatory RNAs; and, most importantly, the turnover of numerous cellular mRNAs in Escherichia coli (5,13,23,33). Homologous RNase E has been identified in more than 50 bacteria, archaea, and plants (28). The 1,061-amino-acid E. coli RNase E protein can be divided into functional portions, from the N terminus to the C terminus, as follows. The N-terminal half (amino acid residues 1 to 529) contains the endonuclease active site (amino acid residues 1 to 395) and a zinc finger region (amino acid residues 400 to 415) (8, 9). The central region includes the membrane anchoring segment A (amino acid residues 565 to 582) and its flanking portions (25) as well as an arginine-rich RNA binding site (amino acid residues 604 to 688) (34). The C-terminal half (amino acid residues 734 to 1061) is an unstructured scaffold domain that contains binding sites for the other core degradosome components, namely, polynucleotide phosphorylase, the RhlB helicase, and the glycolytic enzyme enolase (11,35,45,46,53). Previous studies indicate that the assembled degradosome complex is necessary for normal mRNA degradation and the degradosome components functionally interact during decay of at least some RNAs in E. coli (4,24).The cellular level and activity of RNase E in E. coli are und...

HCCA2 (hepatocellular carcinoma-associated gene 2) was initially identified as a HCC (hepatocellular carcinoma)-specific protein and subsequently, a long splice variant of HCCA2 was identified as a co-activator of transcription factor YY1 (Yin Yang 1). To investigate the role of HCCA2 in HCC genesis and progression, we screened a human fetal liver cDNA library and identified a novel HCCA2-interacting protein, MAD2L2 (MAD2 mitotic arrest deficient-like 2 (yeast)). The interaction between HCCA2 and MAD2L2 was confirmed by in vitro and in vivo binding assays and the interaction domain was mapped to the N-terminus of HCCA2 by sequential deletion. HCCA2 and MAD2L2 also colocalized in the nucleus of Hela cells. Furthermore, overexpression of HCCA2 led to cell cycle arrest at G0/G1 phase and therefore inhibited cell proliferation. Our research suggests that HCCA2 may play a novel role in cell cycle regulation.

The genus Amycolatopsis is well known for its ability to produce antibiotics, and an increasing number of valuable biotechnological applications, such as bioremediation, biodegradation, bioconversion, and potentially biofuel, that use this genus have been developed. Amycolatopsis mediterranei is an industrial-scale producer of the important antibiotic rifamycin, which plays a vital role in antimycobacterial therapy. Genetic studies of Amycolatopsis species have progressed slowly due to the lack of efficient transformation methods and stable plasmid vectors. In A. mediterranei U32, electroporation and replicable plasmid vectors have been developed. Here, we establish a simple and efficient conjugal system by transferring integrative plasmid pDZL802 from ET12567 (pUZ8002) to A. mediterranei U32, with an efficiency of 4 £ 10 ¡5 CFU per recipient cell. This integrative vector, based on the 'BT1 int-attP locus, is a stable and versatile tool for A. mediterranei U32, and it may also be applicable to various other Amycolatopsis species for strain improvement, heterologous protein expression, and synthetic biology experiments.