This review will attempt to cover the present information on the multiple forms of eukaryotic DNA-dependent RNA polymerases, both at the structural and functional level. Nuclear RNA polymerases constitute a group of three large multimeric enzymes, each with a different and complex subunit structure and distinct specificity. The review will include a detailed description of their molecular structure. The current approaches to elucidate subunit function via chemical modification, phosphorylation, enzyme reconstitution, immunological studies, and mutant analysis will be described. In vitro reconstituted systems are available for the accurate transcription of cloned genes coding for rRNA, tRNA, 5 SRNA, and mRNA. These systems will be described with special attention to the cellular factors required for specific transcription. A section on future prospects will address questions concerning the significance of the complex subunit structure of the nuclear enzymes; the organization and regulation of the gene coding for RNA polymerase subunits; the obtention of mutants affected at the level of factors, or RNA polymerases; the mechanism of template recognition by factors and RNA polymerase.

Eukaryotic RNA polymerases.

Factor C* is the component of the RNA polymerase I holoenzyme (factor C) that allows specific transcriptional initiation on a factor D (SL1)- and UBF-activated rRNA gene promoter. The in vitro transcriptional capacity of a preincubated rDNA promoter complex becomes exhausted very rapidly upon initiation of transcription. This is due to the rapid depletion of C* activity. In contrast, C* activity is not unstable in the absence of transcription, even in the presence of nucleoside triphosphates (NTPs). By using 3'dNTPs to specifically halt elongation, C* is seen to remain active through transcription complex assembly, initiation, and the first approximately 37 nucleotides of elongation, but it is inactivated before synthesis proceeds beyond approximately 40 nucleotides. When elongation is halted before this critical distance, the C* remains active and on that template complex, greatly extending the kinetics of transcription and generating manyfold more transcripts than would have been synthesized if elongation had proceeded past the critical distance where C* is inactivated. In complementary in vivo analysis under conditions where C* activity is not replenished, C* activity becomes depleted from cells, but this also occurs only when there is ongoing rDNA transcription. Thus, both in vitro and in vivo, the specific initiation-conferring component of the RNA polymerase I holoenzyme is used stoichiometrically in the transcription process.

Factor C*, the specific initiation component of the mouse RNA polymerase I holoenzyme, is inactivated early in the transcription process.

Effects of androgen and polyamines on the phosphorylation of nucleolar proteins from rat ventral prostates with particular reference to 110-kDa phosphoprotein.

Fresh human semen, diluted 1:1 v/v with 0.15 M NaCl- 0.05 M phosphate, pH 7.5, undergoes a 3-fold increase in total thyrotropin-releasing hormone (TRH) immunoreactivity on incubation at 4 C for 8 to 16 hours. To identify the mechanism for this increase, pooled human semen was incubated at 4, 37, and 60 C, and the change in composition of the immunoreactive TRH peptides was quantitated by high pressure liquid chromatography and radioimmunoassay of TRH. His-Pro diketopiperazine, a biologically active metabolite of TRH consisting of a cyclic dipeptide of histidine and proline, also was measured by specific RIA. The concentration of TRH (pGlu-His-Pro-NH2) dropped precipitously within the first hour after dilution and incubation at all temperatures studied. A hydrophobic TRH-homologous peptide with the amino acid composition (Glu,X,Y,Pro), where X and Y are neutral, nonaromatic amino acids, increased 8-fold during 16 hours of incubation at 4 C. This TRH-homologous peptide is not derived from TRH because it lacks the histidine is not derived from TRH because it lacks the histidine residue. A 3- to 23-fold increase in His-Pro diketopiperazine levels occurred after 4 hours at 37 C. This was not due primarily to enzymatic removal of the pyroglutamyl residue from TRH by pyroglutamate aminopeptidase, since about 1 hour after ejaculation the initial His-Pro diketopiperazine levels were 9.7 +/- 5.1 micrograms/ml, or approximately 1000-fold greater than the corresponding levels of seminal TRH. Because cycloheximide, which blocks ribosomal protein biosynthesis, did not inhibit in vitro production of the TRH-homologous peptide and the His-Pro cyclic dipeptide, these peptides, like TRH, most likely arise from post-translational cleavage and processing from pre-existing macromolecular precursor proteins.

In vitro production of a TRH-homologous peptide and His-Pro diketopiperazine by human semen.

In Vivo Effects of Dexamethasone and Cycloheximide on the Phosphorylation of 110-kDa Proteins and the Protein Kinase Activities of Rat Liver Nucleoli*

Changes in template-engaged and free RNA polymerase I activities in isolated nuclei from rat ventral prostates after treatment with testosterone and cycloheximide.

After having defined reliable assay conditions for free and template-bound RNA polymerase I activities, we examined in detail the effect of thyroid hormone in vivo on nucleolar RNA polymerase I in rat liver. The enzyme activity decreased markedly in the hypothyroid state produced by either thyroidectomy, hypophysectomy or by administration of propylthiouracil (PTU). One large dose (25 microgram/100 g body weight) of T3 given intraperitoneally caused a rapid recovery of the enzyme activity within 24 h either in the presence or absence of the pituitary. The enzyme activity of both free and chromatin-bound enzyme changed in parallel throughout. A similar recovery was noted after daily subcutaneous injections of a physiological dose (1 microgram/100 g body weight) of T3 for about 3 weeks. Analyses of subfractions of the enzyme indicated that only IB-type enzyme (the enzyme fraction containing the whole set of subunits) changed in response to thyroid hormone action, also irrespective of the presence or absence of the pituitary. The possibility that thyroid hormone is a major physiological regulator of the synthesis of RNA polymerase I in rat liver is discussed.

Tetsuya Igarashi

Papers

Changes in template-engaged and free RNA polymerase I activities in isolated nuclei from rat ventral prostates after treatment with testosterone and cycloheximide.

Enhancement by Thyroid Hormone of Free and Bound RNA Polymerase IB Activities in Rat Liver Nucleoli