Mouse and human cell extracts (S100) can support an accurate and efficient transcription initiation on homologous ribosomal RNA gene (rDNA) templates. The cell extracts were fractionated with the aid of a phosphocellulose column into four fractions (termed A, B, C and D), including one containing a major part of the RNA polymerase I activity. Various reconstitution experiments indicate that fraction D is an absolute requirement for the correct and efficient transcription initiation by RNA polymerase I on both mouse and human genes. Fraction B effectively suppresses random initiation on these templates. Fraction A appears to further enhance the transcription which takes place with fractions C and D. Although fractions A, B and C are interchangeable between mouse and human extracts, fraction D is not; i.e. initiation of transcription required the presence of a homologous fraction D for both templates. The factor(s) in fraction D, however, is not literally species-specific, since mouse D fraction is capable of supporting accurate transcription initiation on a rat rDNA template in the presence of all the other fractions from human cell extract under the conditions where human D fraction is unable to support it. We conclude from these experiments that a species-dependent factor in fraction D plays an important role in the initiation of rDNA transcription in each animal species.

The transcription initiation site of the human ribosomal RNA gene (rDNA) was located by using the single-strand specific nuclease protection method and by determining the first nucleotide of the in vitro capped 45S preribosomal RNA. The sequence of 1,211 nucleotides surrounding the initiation site was determined. The sequenced region was found to consist of 75% G and C and to contain a number of short direct and inverted repeats and palindromes. By comparison of the corresponding initiation regions of three mammalian species, several conserved sequences were found upstream and downstream from the transcription starting point. Two short A+T-rich sequences are present on human, mouse, and rat ribosomal RNA genes between the initiation site and 40 nucleotides upstream, and a C+T cluster is located at a position around -60. At and downstream from the initiation site a common sequence, T-4-C-T-G-A-C-A-C-G-C-T-G-T-C-C-T-C-T, was found in the three genes from position -1 through + 18. The strong conservation of these sequences suggests their functional significance in rDNA. The SI nuclease protection experiments with cloned rDNA fragments indicated the presence in human 45S RNA ofmolecules several hundred nucleotides shorter than the supposed primary transcript. The first 19 nucleotides ofthese.molecules appear identical-except for one mismatch-to the nucleotide sequence of the 5' end of a supposed early processing product of the mouse 45S RNA.The ribosomal RNA genes (rDNA) in eukaryotes are transcribed by RNA polymerase I (RNA nucleotidyltransferase, EC 2.7.7.6). This enzyme is in many respects different from the other two RNA polymerases present in eukaryotic cells (1, 2). The regulation of transcription initiation by RNA polymerase II and III is beginning to be understood. Conserved short sequences at fixed distances from the initiation site were found in bacterial genes (3) and in eukaryotic genes transcribed by RNA polymerase II (4, 5), although their function may not be necessarily the same (6, 7). Very limited conserved sequences were found in the prelude region of genes transcribed by RNA polymerase III (8-10). Rather; sequences downstream from the initiation point were found to be needed for correct initiation (11,12).Contrary to polymerases II and III, virtually nothing is known about the regulatory sequences of transcription by RNA polymerase I. Nucleotide sequences of ribosomal RNA transcription initiation regions were determined in several eukaryotes (13)(14)(15)(16)(17)(18)(19). Only very limited similarity was found in those regions of the compared genes where some regulatory sequences might exist (19).In the present work, we have determined the transcription initiation site of the human rRNA gene and then compared the surrounding nucleotide sequences with those ofthe mouse and rat ribosomal genes that were determined previously (16,20,21 (Yamasa Shoyu, Choshi). Rat liver guanylyltransferase was purified as described (22).Preparation of 45S RNA. 45S RNA was prepared from a human mammary tumor c...

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