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BBA - Gene Structure and Expression (v.1727, #2)
The Terminal acidic SANT 1 ( Tacs1) gene of maize is expressed in tissues containing meristems and encodes an acidic SANT domain similar to some chromatin-remodeling complex proteins by Calin O. Marian; Hank W. Bass (pp. 81-86).
While screening for plant homologs of telomeric-complex proteins, we isolated a cDNA for the Terminal acidic SANT 1 ( Tacs1) gene of maize, encoding a 45-kDa protein with a C-terminal Myb/SANT-like domain. Gene expression and protein modeling data indicate that the TACS1 protein may function in chromatin remodeling within shoot primordia or other meristem-containing tissues.
Keywords: Abbreviations; BAC; bacterial artificial chromosome; DBD; DNA-binding domain; SANT; SWI3, ADA2, N-COR, and TFIIIB″; EST; expressed sequence tag; ORF; open reading frame; UTR; untranslated regionTelomere; Myb-like DNA-binding domain; SANT domain; Primordium; Surface electrostatic potential; Plant
The YrdC protein—a putative ribosome maturation factor by Magdalena Kaczanowska; Monica Rydén-Aulin (pp. 87-96).
Release factor one (RF1) terminates protein synthesis in response to stop codons UAG and UAA. A mutant allele of RF1 causes temperature sensitive growth at 42 °C. We have earlier described the isolation of a suppressor of the temperature sensitive phenotype. The suppressor mutation is a small deletion in the open reading frame yrdC, and we have shown that the Δ yrdC mutation leads to immature 30S subunits and, as a consequence, to fewer translating ribosomes. YrdC is a small conserved protein with a dsRNA-binding surface. Here, we have characterized the YrdC protein. We show that the deletion leads to no production of functional protein, and we have indications that the YrdC protein might be essential in a wild type background. The protein is needed for the maturation of 16S rRNA, even though it does not interact tightly with either of the ribosomal subunits, or the 70S particles. The less effective maturation of rRNA affects the ribosomal feedback control, leading to an increase in expression from P1rrnB. We suggest that the function of the YrdC protein is to keep an rRNA structure needed for proper processing of 16S rRNA, especially at lower temperatures. This activity may require other factor(s). We suggest the gene be renamed rimN, and the mutant allele rimN141.
Keywords: Release factor one; 17S rRNA; Ribosome biogenesis; RimN; YciO; SUA5
Transcription arrest caused by long nascent RNA chains by Thomas Bentin; Dmitry Cherny; H. Jakob Larsen; Peter E. Nielsen (pp. 97-105).
The transcription process is highly processive. However, specific sequence elements encoded in the nascent RNA may signal transcription pausing and/or termination. We find that under certain conditions nascent RNA chains can have a strong and apparently sequence-independent inhibitory effect on transcription. Using phage T3 RNA polymerase (T3 RNAP) and covalently closed circular (cccDNA) DNA templates that did not contain any strong termination signal, transcription was severely inhibited after a short period of time. Less than ∼10% residual transcriptional activity remained after 10 min of incubation. The addition of RNase A almost fully restored transcription in a dose dependent manner. Throughout RNase A rescue, an elongation rate of ∼170 nt/s was maintained and this velocity was independent of RNA transcript length, at least up to 6 kb. Instead, RNase A rescue increased the number of active elongation complexes. Thus transcription behaved as an all-or-none process. The mechanism of transcription inhibition was explored using electron microscopy and further biochemical experiments. The data suggest that multiple mechanisms may contribute to the observed effects. Part of the inhibition can be ascribed to the formation of R-loops between the nascent RNA and the DNA template, which provides “roadblocks� to trailing T3 RNAPs. Based on available literature we discuss possible in vivo implications of the results.
Keywords: Abbreviations; RNAP; RNA polymerase; EC; elongation complex; NTPs; ribonucleotide triphosphates; cccDNA; covalently closed circular DNARNA polymerase; RNase; DNA topology; R-loop
Identification of two candidate collecting duct cell-specific cis-acting elements in the Hoxb-7 promoter region by Emmanuelle Plaisier; David Ribes; Pierre Ronco; Jérome Rossert (pp. 106-115).
HOX genes encode highly conserved transcription factors responsible for developmental patterning and postnatal tissue homeostasis. Previous studies have shown that a 1.4-kb segment of the Hoxb-7 proximal promoter drives renal expression of reporter genes specifically in the ureteric bud and collecting ducts. In this study using stably transfected renal tubule cell lines, we have identified three short cis-acting sequences within this promoter segment that cooperate to induce high-level expression specifically in collecting duct cells. In addition to an inverted CCAAT box (−71/−67) that acts as an ubiquitous enhancer and binds the transcription factor CBF/NF-Y, two different cis-acting sequences, named CDSE-1 and CDSE-2 (for Collecting Duct Specific Element 1 and 2), allow collecting duct cell-specific promoter activation. CDSE-1 (−56/−34) is composed of two E-boxes separated by a 9-bp GC-rich sequence. Only the latter sequence enhances reporter gene expression specifically in collecting duct cells. CDSE-2 (−34/−13) contains sequence bears high homology with a segment of the Pax-2 promoter. CDSE-2 also conveys cell specificity but has no enhancer activity by itself.
Keywords: Hoxb7; Promoter; CCAAT box; USF; Collecting duct; Kidney
Deconstructing PTI-1: PT1-1 is a truncated, but not mutated, form of translation elongation factor 1A1, eEF1A1 by Francisco Mansilla; Lise Lotte Hansen; Helle Jakobsen; Niels Ole Kjeldgaard; Brian F.C. Clark; Charlotte R. Knudsen (pp. 116-124).
The prostate tumor-inducing gene 1 ( PTI-1) transcript is detected in various human carcinoma cells. PTI-1 is reported to consist of a 5′ untranslated region (5′ UTR) homologous to mycoplasma 23S rRNA and a coding region corresponding to a truncated and mutated form of the translation elongation factor 1A, eEF1A. We have found that the PTI-1 transcript may encode a truncated, but not mutated, form of the human isoform eEF1A1. Additionally, the 5′ UTR sequence of PTI-1 from genomic DNA of different cell lines and blood samples varies from the original sequence. This 5′-UTR region of PTI-1 presents a fusion of E. coli and Mycoplasma hyorhinis 23S rRNA. We have overexpressed the potential PTI-1 protein in E. coli and various human cell lines. The resulting protein could be detected by western blotting using anti-eEF1A antibodies. However, we were unable to detect the PTI-1 protein in LNCaP cell extracts. The potential roles of the PTI-1 protein in carcinogenesis and the origin of the PTI-1 gene in the human genome are discussed.
Keywords: eEF1A1; PTI-1 protein; PTI-1 mRNA; Prostate cancer
Transcriptional regulation of FKLF-2 (KLF13) gene in erythroid cells by Ayako Mitsuma; Haruhiko Asano; Tomohiro Kinoshita; Takashi Murate; Hidehiko Saito; George Stamatoyannopoulos; Tomoki Naoe (pp. 125-133).
FKLF-2 (KLF13) was cloned from fetal globin-expressing tissues and has been shown to be abundantly expressed in erythroid cells. In this study we examined the transcriptional regulation of the KLF13 gene. A 5.5 kb 5′ flanking region cloned from mouse erythroleukemia (MEL) cell genomic DNA showed that major cis regulatory activities exist in the 550 bp sequence to the unique transcription start site, and that the promoter is more active in K562 cells than in COS-7 cells. The promoter was trans-activated by co-expressed GATA-1 through the sequence containing two CCAAT motifs, suggesting that GATA-1 is involved in the abundant expression of KLF13 mRNA in the erythroid tissue. Dual action, i.e. activating effect in COS-7 and repressive effect in K562 cell, was observed on its own promoter, suggesting a feedback mechanism for the transcriptional control of the KLF13 gene in the erythroid environment. These findings provide an insight on the mechanism of inducible mRNA expression of the KLF13 gene in erythroid cells.
Keywords: Erythroid differentiation; KLF family; Promoter; Transcriptional regulation
Human ribosomal protein S26 suppresses the splicing of its pre-mRNA by Anton V. Ivanov; Alexey A. Malygin; Galina G. Karpova (pp. 134-140).
Human recombinant ribosomal protein S26 (rpS26) was shown to interact with its pre-mRNA intron I and mRNA fragment. Endogenous rpS26 in HeLa nuclear extract was also found to bind to the intron I, and with a lower extent to the mRNA fragment. The addition of recombinant rpS26 to the nuclear extract increased the binding largely. The in vitro splicing of an RNA that contained exon I, intron I and part of exon II of the rpS26 pre-mRNA yielded conventional and alternative mRNAs. Recombinant rpS26 was found to suppress the formation of both mRNAs. Sites of the pre-mRNA involved in the binding to rpS26 were detected by toe-printing. Nucleotides that caused a stop (pause) of the reverse transcription formed two clusters on the RNA secondary structure. One cluster including A69, A287 and A303 arranged the conventional 3′ site of splicing, another one including A131, A136, G156, A166 and A264 arranged the alternative 3′ site of splicing.
Keywords: Human ribosomal protein S26; RNA protein binding; Conventional and alternative splicing; Regulation of splicing
Transcription factor NF2d9 (LBP-1a) interacts with the positive regulatory element for the xenobiotic responsive element by Kouichi Kurose; Masahiro Tohkin; Ryuichi Hasegawa (pp. 141-144).
PREX is a positive regulatory element for xenobiotic responsive element (XRE)-mediated gene expression that is located upstream of the XRE in the CYP2A8 gene. Using gel mobility shift assays, we demonstrated that NF2d9 (LBP-1a), a transcription factor related to CP2 (LBP-1c/LSF), bound directly to PREX and also interacts indirectly with XRE. Luciferase-reporter gene assays showed that the overexpression of NF2d9 enhanced PREX and XRE-driven CYP2A8 gene transcriptional induction. These findings suggest that the interaction of NF2d9 with PREX and XRE enhances XRE-driven CYP2A8 gene transcriptional induction.
Keywords: Abbreviations; 3-MC; 3-methylcholanthrene; AhR; arylhydrocarbon receptor; Arnt; AhR nuclear translocator; PREX; positive regulatory element for XRE-mediated gene expression; XRE; xenobiotic responsive elementArylhydrocarbon receptor; CYP2A8; NF2d9; PREX; Xenobiotic responsive element
A novel isoform of Cbl-associated protein that binds protein kinase A by Sarah A. Matson; Genevieve C. Pare; Michael S. Kapiloff (pp. 145-149).
A novel isoform of Cbl-associated protein (CAP) was identified in a yeast two-hybrid screen for A-kinase anchoring proteins expressed in the heart. CAP is a scaffold protein implicated in insulin signaling and cytoskeleton regulation. The protein kinase A binding site is encoded by a previously unidentified, alternatively spliced exon.
Keywords: A-kinase anchoring protein; Protein kinase A; Cbl-associated protein; Heart; SORBS1