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BBA - Gene Regulatory Mechanisms (v.1829, #2)
The RNA polymerase bridge helix YFI motif in catalysis, fidelity and translocation by Yuri A. Nedialkov; Kristopher Opron; Fadi Assaf; Irina Artsimovitch; Maria L. Kireeva; Mikhail Kashlev; Robert I. Cukier; Evgeny Nudler; Zachary F. Burton (pp. 187-198).
The bridge α-helix in the β′ subunit of RNA polymerase (RNAP) borders the active site and may have roles in catalysis and translocation. In Escherichia coli RNAP, a bulky hydrophobic segment near the N-terminal end of the bridge helix is identified (β′ 772-YFI-774; the YFI motif). YFI is located at a distance from the active center and adjacent to a glycine hinge (β′ 778-GARKG-782) involved in dynamic bending of the bridge helix. Remarkably, amino acid substitutions in YFI significantly alter intrinsic termination, pausing, fidelity and translocation of RNAP. F773V RNAP largely ignores the λ tR2 terminator at 200μM NTPs and is strongly reduced in λ tR2 recognition at 1μM NTPs. F773V alters RNAP pausing and backtracking and favors misincorporation. By contrast, the adjacent Y772A substitution increases fidelity and exhibits other transcriptional defects generally opposite to those of F773V. All atom molecular dynamics simulation revealed two separate functional connections emanating from YFI explaining the distinct effects of substitutions: Y772 communicates with the active site through the link domain in the β subunit, whereas F773 communicates through the fork domain in the β subunit. I774 interacts with the F-loop, which also contacts the glycine hinge of the bridge helix. These results identified negative and positive circuits coupled at YFI and employed for regulation of catalysis, elongation, termination and translocation.► The bridge helix contains a bendable glycine hinge. ► Adjacent bulky hydrophobic residues and surrounding protein regulate the hinge. ► Mutations in these bulky residues modify transcription elongation. ► Pausing, catalysis, fidelity, termination and translocation are affected.
Keywords: Abbreviations; Ec; Escherichia coli; Sc; Saccharomyces cerevisiae; Mj; Methanocaldococcus jannaschii; Tt; Thermus thermophilus; exonuclease III; Exo III; GMPcPP; α,β-methylene guanosine triphosphate; NTP; nucleoside triphosphate; PPi; pyrophosphate (diphosphate); RNAP; RNA polymerase; RNA G9; 9 nucleotide RNA ending in 3′-GMP; TEC; ternary elongation complex (RNAP, DNA, nascent RNA)RNA polymerase; Bridge helix; Termination; Pausing; Transcriptional fidelity; Translocation
PICKLE is a CHD subfamily II ATP-dependent chromatin remodeling factor by Kwok Ki Ho; Heng Zhang; Barbara L. Golden; Joe Ogas (pp. 199-210).
PICKLE plays a critical role in repression of genes that regulate development identity in Arabidopsis thaliana. PICKLE codes for a putative ATP-dependent chromatin remodeler that exhibits sequence similarity to members of subfamily II of animal CHD remodelers, which includes remodelers such as CHD3/Mi-2 that also restrict expression of developmental regulators. Whereas animal CHD3 remodelers are a component of the Mi-2/NuRD complex that promotes histone deacetylation, PICKLE promotes trimethylation of histone H3 lysine 27 suggesting that it acts via a distinct epigenetic pathway. Here, we examine whether PICKLE is also a member of a multisubunit complex and characterize the biochemical properties of recombinant PICKLE protein. Phylogenetic analysis indicates that PICKLE-related proteins in plants share a common ancestor with members of subfamily II of animal CHD remodelers. Biochemical characterization of PICKLE in planta, however, reveals that PICKLE primarily exists as a monomer. Recombinant PICKLE protein is an ATPase that is stimulated by ssDNA and mononucleosomes and binds to both naked DNA and mononucleosomes. Furthermore, recombinant PICKLE exhibits ATP-dependent chromatin remodeling activity. These studies demonstrate that subfamily II CHD proteins in plants, such as PICKLE, retain ATP-dependent chromatin remodeling activity but act through a mechanism that does not involve the ubiquitous Mi-2/NuRD complex.► PICKLE-related proteins share a common ancestor with CHD3 chromatin remodelers. ► Homology modeling predicts that PICKLE has a SANT-SLIDE DNA-binding domain. ► Unlike animal CHD3 proteins, PICKLE primarily exists as a monomer. ► Recombinant PICKLE is an ATPase that is stimulated by ssDNA and mononucleosomes. ► Recombinant PICKLE exhibits ATP‐dependent chromatin remodeling activity.
Keywords: Abbreviations; PHD; plant homeodomain; PKL; PICKLE; HDAC; histone deacetylase; MBD2; methyl-CpG-binding domain protein 2; H3K27me3; trimethylation of lysine 27 histone H3; LEC; Leafy Cotyledon; ACT7; Actin7; UBQ10; Ubiquitin10; PKR; Pickle-related; DUF; domain of unknown function; ssDNA; single-stranded DNA; dsDNA; double-stranded DNA WT, wild type; PRC2; Polycomb repressive complex 2Chromatin remodeling; CHD protein; ATP-dependent chromatin remodeler; SANT-SLIDE; Developmental identity
The mechanism of alternative splicing of the X-linked NDUFB11 gene of the respiratory chain complex I, impact of rotenone treatment in neuroblastoma cells by Damiano Panelli; Francesca Paola Lorusso; Francesco Papa; Patrizio Panelli; Alessandro Stella; Massimo Caputi; Anna Maria Sardanelli; Sergio Papa (pp. 211-218).
A study is presented on the regulation of alternative splicing (AS) of the Ndufb11 gene of complex I of the mitochondrial respiratory chain and the impact on this process of rotenone treatment in neuroblastoma cells. In physiological conditions the Ndufb11 gene produces at high level a short transcript isoform encoding for a 153 aa protein. This subunit is essential for the assembly of a functional and stable mammalian complex I. The gene produces also, at low level, a longer transcript isoform encoding for a 163 aa protein whose role is unknown. Evidence is presented here showing that the level of the two isoforms is regulated by three DGGGD ESS elements located in exon 2 which can bind the hnRNPH1 protein. In neuronal cells rotenone treatment affects the Ndufb11 alternative splicing pathway, with the increase of the 163/153 mRNAs ratio. This effect appears to be due to the down-regulation of the hnRNPH1 protein. Since rotenone induces apoptosis in neuronal cells, the post-transcriptional regulation of the Ndufb11 gene can be involved in the programmed cell death process.► The regulation of alternative splicing of the Ndufb11 gene is presented. ► The level of the two Ndufb11 alternative isoforms is regulated by three ESS. ► Rotenone treatment alters the Ndufb11 alternative splicing pathway. ► This effect appears to be due to the down-regulation of the hnRNPH1 protein.
Keywords: Alternative splicing; Exonic splicing silencers; Respiratory complex I
Characterization of the transcriptional stimulatory properties of the Pseudomonas putida RapA protein by Ewa Stec-Dziedzic; Łyzen Robert Łyżeń; Skarfstad Eleonore Skärfstad; Victoria Shingler; Agnieszka Szalewska-Pałasz (pp. 219-230).
RNA polymerase-associated factors can significantly affect its performance at specific promoters. Here we identified a Pseudomonas putida RNA polymerases-associated protein as a homolog of Escherichia coli RapA. We found that P. putida RapA stimulates the transcription from promoters dependent on a variety of σ-factors (σ70, σS, σ54, σ32, σE) in vitro. The level of stimulation varied from 2- to 10-fold, with the maximal effect observed with the σE-dependent PhtrA promoter. Stimulation by RapA was apparent in the multi-round reactions and was modulated by salt concentration in vitro. However, in contrast to findings with E. coli RapA, P. putida RapA-mediated stimulation of transcription was also evident using linear templates. These properties of P. putida RapA were apparent using either E. coli- or P. putida-derived RNA polymerases. Analysis of individual steps of transcription revealed that P. putida RapA enhances the stability of competitor-resistant open-complexes formed by RNA polymerase at promoters. In vivo, P. putida RapA can complement the inhibitory effect of high salt on growth of an E. coli RapA null strain. However, a P. putida RapA null mutant was not sensitive to high salt. The in vivo effects of lack of RapA were only detectable for the σE-PhtrA promoter where the RapA-deficiency resulted in lower activity. The presented characteristics of P. putida RapA indicate that its functions may extend beyond a role in facilitating RNA polymerase recycling to include a role in transcription initiation efficiency. ► Pseudomonas putida RapA is a core RNA polymerase-associated protein. ► P. putida RapA activates transcription in multi-round reactions. ► P. putida RapA stimulates transcription dependent on various σ factors. ► σE-pHtrA promoter activity is impaired in P. putida rapA mutant. ► P. putida RapA enhances stability of competitor resistant open complexes.
Keywords: Abbreviations; IPTG; isopropyl β-; d; -1-thiogalactopyranoside; RNAP; RNA polymeraseRapA; Pseudomonas putida; Transcription factors; RNA polymerase
Neuron-restrictive silencer factor functions to suppress Sp1-mediated transactivation of human secretin receptor gene by Yuan Yuan; Billy K.C. Chow; Vien H.Y. Lee; Leo T.O. Lee (pp. 231-238).
In the present study, a functional neuron restrictive silencer element (NRSE) was initially identified in the 5′ flanking region (−83 to −67, relative to ATG) of human secretin receptor (hSCTR) gene by promoter assays coupled with scanning mutation analyses. The interaction of neuron restrictive silencer factor (NRSF) with this motif was later indicated via gel mobility shift and ChIP assays. The silencing activity of NRSF was confirmed by over-expression and also by shRNA knock-down of endogenous NRSF. These studies showed an inverse relationship between the expression levels of NRSF and hSCTR in the cells. As hSCTR gene was previously shown to be controlled by two GC-boxes which are regulated by the ratio of Sp1 to Sp3, in the present study, the functional interactions of NRSF and Sp proteins to regulate hSCTR gene was investigated. By co-immunoprecipitation assays, we found that NRSF could be co-precipitated with Sp1 as well as Sp3 in PANC-1 cells. Interestingly, co-expressions of these factors showed that NRSF could suppress Sp1-mediated, but not Sp3-mediated, transactivation of hSCTR. Taken together, we propose here that the down-regulatory effects of NRSF on hSCTR gene expression are mediated via its suppression on Sp1-mediated transactivation.► The binding of NRSF to hSCTR was investigated by gel shift and ChIP assays. ► The interactions of NRSF with Sp1 and Sp3 were studied by co-immunoprecipitation. ► The Sp1 to Sp3 ratio mediates the silencing effects of NRSF on hSCTR. ► Treatment of TSA relieves”” that HDAC is key to the silencer activity of NRSF.
Keywords: Abbreviations; ChIP; chromatin immunoprecipitation; hSCTR; human secretin receptor; NRSF; neuron-restrictive silencer factor; NRSE; neuron-restrictive silencer element; PANC-1; human pancreatic ductile carcinoma; shRNA; short hairpin RNA; TSA; Trihostatin ASecretin receptor; Neuron restrictive silencer factor; Sp-protein; Transcriptional regulation
Downregulation of miR-145 associated with cancer progression and VEGF transcriptional activation by targeting N-RAS and IRS1 by Yu Yin; Zhi-Ping Yan; Na-Na Lu; Qing Xu; Jun He; Xu Qian; Jing Yu; Xin Guan; Bing-Hua Jiang; Ling-Zhi Liu (pp. 239-247).
MicroRNA-145 (miR-145) is downregulated in various tumor types. However, its mechanism in inhibiting tumor growth and angiogenesis remains to be elucidated. In this study, we found that miR-145 was significantly downregulated in the plasma and cancer tumor tissues of colorectal cancer (CRC) patients, and overexpression of miR-145 inhibited cell proliferation, migration and invasion. To understand the potential mechanism of miR-145 in inhibiting tumor growth, we showed that miR-145 blocked the activation of AKT and ERK1/2 pathways, and the expression of HIF-1 and VEGF via directly targeting N-RAS and IRS1, and VEGF is an important effector for tumor growth. Forced expression of N-RAS and IRS1 restored VEGF expression via transcriptional activation. MiR-145 also inhibited N-RAS and IRS1 expression to suppress AKT and ERK1/2 activation, and VEGF expression in mouse xenograft tumors. To test the clinical relevance of these results, we used 60 pairs of colorectal cancer tissues and adjacent normal tissues, analyzed the levels of miR-145, N-RAS and IRS1 expression in these tissues, and found that miR-145 levels were significantly inversely correlated with N-RAS and IRS1 levels in these colorectal cancer tissues, suggesting the important implication of our findings in translational application for colorectal cancer diagnostics and treatment in the future.► Circulating miR-145 is decreased in the plasma of colorectal cancer patients. ► MiR-145 is downregulated with inverse correlation with N-RAS and IRS1 levels in cancer stages. ► MiR-145 inhibits AKT/ERK/HIF-1/VEGF pathway by directly targeting N-RAS and IRS1. ► The study indicates that miR-145 levels can be used as a new biomarker for cancer diagnosis.
Keywords: Abbreviations; CRC; colorectal cancer; UTR; untranslated region; IRS1; insulin receptor substrate-1; IGF-IR; type 1 insulin-like growth factor receptor; FBS; fetal bovine serum; VEGF; vascular endothelial growth factor; SW1116-NC; SW1116 stably expressing negative control of miRNA precursor; SW1116-miR-145; SW1116 stably expressing miR-145; IHC; immunohistochemistry; ISH; in situ; hybridizationmiR-145; N-RAS; IRS1; VEGF; Colorectal cancer; Angiogenesis