Structure of a transcribing T7 RNA polymerase initiation complex

The structure of a T7 RNA polymerase (T7 RNAP) initiation complex captured transcribing a trinucleotide of RNA from a 17-base pair promoter DNA containing a 5-nucleotide single-strand template extension was determined at a resolution of 2.4 angstroms. Binding of the upstream duplex portion of the promoter occurs in the same manner as that in the open promoter complex, but the single-stranded template is repositioned to place the +4 base at the catalytic active site. Thus, synthesis of RNA in the initiation phase leads to accumulation or "scrunching" of the template in the enclosed active site pocket of T7 RNAP. Only three base pairs of heteroduplex are formed before the RNA peels off the template.     

稳定表达T_7RNA聚合酶的猪睾丸细胞系的建立

根据GenBank中登录的T7RNA聚合酶基因参考序列,设计合成了1对特异性引物扩增对T7RNA聚合酶基因进行扩增,将测序正确的T7RNA聚合酶基因和真核表达载体pIRES2-EGFP双酶切后进行连接构建pIRES2-EGFP-T7RNA RNA质粒。再将构建正确的pIRES2-EGFP-T7RNA质粒经用脂质体法转染猪睾丸细胞,通过G418筛选和单细胞克隆化,同时构建pET-32a-RED原核表达质粒载体,用其检测T7启动子控制下的红色荧光蛋白的表达。结果表明,建立的ST/T7RNA细胞系经20次传代仍然能稳定表达T7RNA聚合酶。结果显示,成功建立能稳定表达T7RNA聚合酶的猪睾丸细胞系,为猪瘟病毒反向遗传操作平台奠定了基础。     

Abortive initiation and productive initiation by RNA polymerase involve DNA scrunching

Using single-molecule DNA nanomanipulation, we show that abortive initiation involves DNA "scrunching"--in which RNA polymerase (RNAP) remains stationary and unwinds and pulls downstream DNA into itself--and that scrunching requires RNA synthesis and depends on RNA length. We show further that promoter escape involves scrunching, and that scrunching occurs in most or all instances of promoter escape. Our results support the existence of an obligatory stressed intermediate, with approximately one turn of additional DNA unwinding, in escape and are consistent with the proposal that stress in this intermediate provides the driving force to break RNAP-promoter and RNAP-initiation-factor interactions in escape.     

Detergent-solubilized RNA polymerase from cells infected with foot-and-mouth disease virus

The foot-and-mouth disease virus RNA polymerase complex was dissociated from cellular membranes with deoxycholate in the presence of dextran sulfate. The soluble polymerase complex was active in the cell-free synthesis of virus-specific RNA; solubilization of the complex permitted direct analysis of the cell-free reaction mixtures without recourse to RNA extraction. A major RNA-containing component found early during cell-free incubation ranged from approximately 140 to 300S. The final major products of the cell-free system were 37S virus RNA, 20S ribonuclease-resistant RNA, and a 50S component containing RNA.     

Crystal structure of the eukaryotic 60S ribosomal subunit in complex with initiation factor 6

Protein synthesis in all organisms is catalyzed by ribosomes. In comparison to their prokaryotic counterparts, eukaryotic ribosomes are considerably larger and are subject to more complex regulation. The large ribosomal subunit (60S) catalyzes peptide bond formation and contains the nascent polypeptide exit tunnel. We present the structure of the 60S ribosomal subunit from Tetrahymena thermophila in complex with eukaryotic initiation factor 6 (eIF6), cocrystallized with the antibiotic cycloheximide (a eukaryotic-specific inhibitor of protein synthesis), at a resolution of 3.5 angstroms. The structure illustrates the complex functional architecture of the eukaryotic 60S subunit, which comprises an intricate network of interactions between eukaryotic-specific ribosomal protein features and RNA expansion segments. It reveals the roles of eukaryotic ribosomal protein elements in the stabilization of the active site and the extent of eukaryotic-specific differences in other functional regions of the subunit. Furthermore, it elucidates the molecular basis of the interaction with eIF6 and provides a structural framework for further studies of ribosome-associated diseases and the role of the 60S subunit in the initiation of protein synthesis.     

Two domains of yeast U6 small nuclear RNA required for both steps of nuclear precursor messenger RNA splicing

U6 is one of the five small nuclear RNA's (snRNA's) that are required for splicing of nuclear precursor messenger RNA (pre-mRNA). The size and sequence of U6 RNA are conserved among organisms as diverse as yeast and man, and so it has been proposed that U6 RNA functions as a catalytic element in splicing. A procedure for in vitro reconstitution of functional yeast U6 small nuclear ribonucleoproteins (snRNP's) with synthetic U6 RNA was applied in an attempt to elucidate the function of yeast U6 RNA. Two domains in U6 RNA were identified, each of which is required for in vitro splicing. Single nucleotide substitutions in these two domains block splicing either at the first or the second step. Invariably, U6 RNA mutants that block the first step of splicing do not enter the spliceosome. On the other hand, those that block the second step of splicing form a spliceosome but block cleavage at the 3' splice site of the intron. In both domains, the positions of base changes that block the second step of splicing correspond exactly to the site of insertion of pre-mRNA-type introns into the U6 gene of two yeast species, providing a possible explanation for the mechanism of how these introns originated and adding further evidence for the proposed catalytic role of U6 RNA.     

Visualizing the higher order folding of a catalytic RNA molecule

The higher order folding process of the catalytic RNA derived from the self-splicing intron of Tetrahymena thermophila was monitored with the use of Fe(II)-EDTA-induced free radical chemistry. The overall tertiary structure of the RNA molecule forms cooperatively with the uptake of at least three magnesium ions. Local folding transitions display different metal ion dependencies, suggesting that the RNA tertiary structure assembles through a specific folding intermediate before the catalytic core is formed. Enzymatic activity, assayed with an RNA substrate that is complementary to the catalytic RNA active site, coincides with the cooperative structural transition. The higher order RNA foldings produced by Mg(II), Ca(II), and Sr(II) are similar; however, only the Mg(II)-stabilized RNA is catalytically active. Thus, these results directly demonstrate that divalent metal ions participate in general folding of the ribozyme tertiary structure, and further indicate a more specific involvement of Mg(II) in catalysis.     

Unexpectedly high levels of HIV-1 RNA and protein synthesis in a cytocidal infection

The expression of a laboratory strain of HIV-1 (HTLV-IIIB) has been studied in mitogen-stimulated peripheral blood lymphocytes (PBLs) and in two lymphoid cell lines (CEM cells and C8166 cells). HIV-expressing cells contained from 300,000 to 2,500,000 copies of viral RNA per cell. Near-synchronous expression of an active infection could be achieved in C8166 cells. In these cells, the high copy numbers of viral RNA used as much as 40% of total protein synthesis for the production of viral gag protein, with high levels of viral RNA and protein synthesis preceding cell death by 2 to 4 days.