R G Roeder

Selective and accurate initiation of transcription at the ad2 major late promotor in a soluble system dependent on purified rna polymerase ii and dna

Transcription of Ad2 DNA templates in the presence of crude cellular extracts supplemented with exogenous (purified) RNA polymerase II is selectively and accurately initiated at the major late viral promoter at map position 16.45. Specific initiation has been demonstrated by a combination of hybridization, nuclease S1 mapping, size and partial sequence (fingerprint) analyses of the transcripts generated with various templates. With intact Ad2 DNA, transcription is terminated ell before the end of the 28 kb transcription unit is reached. With truncated templates (which contain intact promoter regions and several hundred base pair segments of the transcribed region) the expected run-off products are observed, along with a low level of prematurely terminated transcripts. The 560 nucleotide run-off product of the Sma l-f template (coordinates 11.6-18.2) was shown to contain all the large RNAase T1 oligonuc eotides that are characteristic of the corresponding in vivo transcript from this region; in addition, the 5 terminal undecanucleotide appears to be both capped and methylated. We have investigated various parameters (salt, metal ion and template concentrations) that affect the level of specific transcription in the crude system and have found that, under optimal conditions, specific transcription of Ad2 DNA continues for several hours. In addition, specific transcription initiation at the late promoter is observed with extracts derived from either virus-infected or uninfected KB cells and with class II RNA polymerases isolated from either human calf, murine or amphibian cells. RNA polymerase II from wheat germ does not function in this system.

Association of a 5S gene transcription factor with 5S RNA and altered levels of the factor during cell differentiation.

We have previously purified from Xenopus ovaries a protein factor (TF IIIA) which is necessary for the accurate in vitro transcription of 5S RNA genes. We now report that this factor (a 5S gene transcription effector) is identical by immunological, chemical and functional criteria to the protein associated with 5S RNA (the gene product) as a 7S ribonucleoprotein (RNP) complex in immature oocytes. After oocyte maturation, TF IIIA is no longer detectable functionally or immunologically in unfertilized eggs, which do not synthesize 5S RNA in vitro. Moreover, we cannot detect TF IIIA immunologically in extracts fron Xenopus somatic cells which, nevertheless, efficiently transcribe 5S genes.

Low molecular weight viral RNAs transcribed by RNA polymerase III during adenovirus 2 infection

Nuclei isolated from human cells productively infected with adenovirus 2 have been shown to synthesize four low molecular weight RNA species which hybridize efficiently to viral DNA. One species corresponds to the 5.5S or VA RNA (Ohe, Weissman, and Cooke, 1969), and is designated V156. The other three species are novel and have been designated V200, V140, V130, since they are approximately 200, 140, and 130 nucleotides in length, respectively. These viral RNAs retain their distinct electrophoretic properties after denaturation with formamide. RNA species with electrophoretic mobilities similar to those of the V200, V156, and V140 RNAs have been found in the cytoplasmic fraction of cells at late times after adenovirus infection. In isolated nuclei, the V200, V156, V140, and V130 RNAs are all synthesized by DNA-dependent RNA polymerase III, since synthesis is sensitive to high but not to low concentrations of alpha-amanitin. The synthesis of these low molecular weight RNAs continues for a prolonged period of time in isolated nuclei, suggesting that reinitiation occurs. Adenovirus 2 DNA fragments obtained by digestion with restriction endonucleases Eco RI and Sma I were used to map the location of the DNA sequences which encode the RNAs. All the low molecular weight RNAs hybridized to a region of the genome between o.18 and 0.38 fractional lengths from the left end of the adenovirus genome, suggesting that the respective DNA sequences are clustered. Other nonviral low molecular weight RNAs are synthesized in nuclei isolated from infected cells. These include the cellular 5S rRNA species which was minitored by its hybridization to purified 5S DNA from Xenopus laevis.

DNA-dependent RNA polymerase levels during the response of human peripheral lymphocytes to phytohemagglutinin

The cellular levels of the various RNA polymerases have been monitored in resting human peripheral lymphocytes and in lymphocytes stimulated by phytohemagglutin. Activity was measured in the presence of exogenous templates following solubilization and chromatographic resolution of the different RNA polymerases. Resting lymphocytes contain Class I, II, and III RNA polymerases, although the respective levels of activity are very low compared to the levels in metabolically active cell types. During the PHA-induced transformation of resting lymphocytes, the Class I, II, and III enzyme levels rise dramatically. During four days exposure to PHA, the levels of RNA polymerases I and III (which synthesize, respectively, rRNA and the transfer and 5S RNAs) increase 17 fold, while the level of RNA polymerase II (which synthezies heterogeneous nuclear RNA) increase 8 fold. The possible relationship between enzyme levels and the regulation of gene expression is discussed.

Transcription of specific genes in isolated nuclei by exogenous RNA polymerases

Mouse plasmacytoma (MOPC) 460 cells contain two chromatographic forms of RNA polymerase III (IIIA and IIIB) in addition to the major class I and II RNA polymerases. Nuclei isolated from these cells actively synthesize RNA. Among the discrete transcription products observed are the 5S and 4.5S RNAs and additional low molecular weight RNA species (approximately 5.8S, 6.3S, and 6.6S in size). The 4.5S RNAs appear to be tRNA precursors since they can be converted in vitro to 4S RNAs. Studies with alpha-amanitin have shown that the synthesis of these discrete RNA species, and other uncharacterized transcripts somewhat larger in size, is mediated by an endogenous RNA polymerase III activity(ies). Nuclear RNA synthesis is stimulated by exogenous purified RNA polymerases. Exogenous MOPC class III RNA polymerases stimulate the synthesis of each of the distinct low molecular weight species (including 5S and 4.5S RNAs) about 3-6 fold. The hybridization of nuclear transcripts to purified 5S genes (5S DNA) confirms that exogenous class III RNA polymerases stimulate (approximately 4 fold) the synthesis of ribosomal 5S RNA. The 5S RNA genes in nuclei are transcribed asymmetrically by both the endogenous and the exogenous class III enzymes. Exogenous RNA polymerase III from Xenopus laevis ovaries stimulates 4.5S and 5S RNA synthesis in MOPC nuclei as effectively as do the MOPC class III RNA polymerases. However, exogenous MOPC class I and II RNA polymerases do not stimulate 4.5S and 5S RNA synthesis, suggesting that this effect is specific for the structurally similar class III RNA polymerases.

Xenopus laevis histone genes: Variant H1 genes are present in different clusters

The Xenopus laevis histone genes have been isolated from a bacteriophage lambda library using a cloned cDNA probe to X. laevis H4 mRNA (pX1ch4). Their structural organization has been determined by restriction mapping, blot hybridization and hybridization selection and translation of Xenopus histone mRNAs. They are clustered and probably tandemly arranged but, in contrast to invertebrate histone genes (Kedes, 1979), there has been extensive sequence divergence in the spacer regions of the clusters. Also, the order of the genes within individual clusters is not conserved. We report the isolation of two variant histone H1 gene and find that the order of the nucleosomal core histone genes within a cluster containing an H1B gene is different from that found in two clusters containing the H1A gene.

SELECTIVE TRANSCRIPTION OF THE 5S RNA GENES IN ISOLATED CHROMATIN BY RNA POLYMERASE III

ABSTRACT Previous studies have shown that the genes which encode the ribosomal 5S RNA and the tRNAs are transcribed by a class III enzyme(s) in somatic cells. To investigate the mechanism and regulation of RNA polymerase III function, the in vitro transcription of the 5S RNA genes expressed in X. laevis oocytes is being analyzed. The RNA polymerase III from these oocytes has been purified to homogeneity and has a complex subunit structure very similar to that of somatic cell class III enzymes. The present studies suggest that the oocyte RNA polymerase III does not accurately transcribe the 5S RNA genes present in a purified recombinant plasmid DNA, even though the 5S DNA may be more efficiently transcribed than the adjacent bacterial plasmid DNA sequences. Strikingly different results were obtained when chromatin from immature X. laevis oocytes, active in 5S RNA synthesis, was used as the template for homologous RNA polymerase III. Although this chromatin contains endogenous RNA polymerase III activity, the level of total RNA synthesis and the level of 5S RNA synthesis were stimulated 10-50 fold by exogenous RNA polymerase III. Furthermore, transcription of the 5S RNA genes present in chromatin was highly selective (3000-fold above random) and predominantly asymmetric. X. laevis oocyte RNA polymerase I stimulated total RNA synthesis from this chromatin about twofold but did not stimulate transcription of the 5S genes. It is concluded that both chromosomal proteins and a purified homologous RNA polymerase III are necessary and sufficient for selective transcription of the 5S RNA genes in vitro .

STRUCTURE, FUNCTION, AND REGULATION OF RNA POLYMERASES IN ANIMAL CELLS

ABSTRACT. The mouse plasmacytoma class I, II, and III RNA polymerases have been purified and shown to have distinct structures and functions. The two large molecular weight subunits and some low molecular weight subunits differ between the class I, II, and III enzymes. However, some small subunits appear common to two or to three enzyme classes. α-Amanitin was used to distinguish the endogenous RNA polymerase activities of the class I, II, and III RNA polymerases in isolated nuclei and to show their functions, respectively, in transcription of the genes for the rRNAs, the HnRNAs, and the transfer and 5S RNAs. The cellular activity levels of solubilized RNA polymerases I and III vary with the physiological state of the cell in different mouse tissues, suggesting that the activities of the genes which they transcribe are regulated in part by specific enzyme levels in adult cell types. The increased RNA polymerase I levels in malignant mouse plasmacytoma cells reflect primarily increased enzyme concentrations. The levels of RNA polymerase II in mouse tissues show much less variability suggesting similar rates of HnRNA synthesis in these cell types or cellular excesses of this enzyme. During very early embryonic development in X. laevis all RNA polymerases are present in vast cellular excess and changes in specific gene function are not accompanied by changes in total enzyme levels, suggesting that factors other than enzyme levels are involved in regulating the transcription of all genes during this developmental period.