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A quantitative dose-response study was made of the inhibition by actinomycin D of the synthesis of various RNA species in cultured L cells. After a suitable

The promoters of several eukaryotic genes transcribed by RNA polymerase H contain elements located downstream of the transcriptional start site. To gain insight into how these elements function in the formation of an active transcription complex, we have cloned and sequenced the cDNA that encodes 8, a protein that binds to critical downstream promoter elements in the mouse ribosomal protein rpL30 and rpL32 genes. Our results revealed that the 6 protein contains four C-terminal zinc fingers, which are essential for its DNA binding capability and a very unusual N-terminal domain that includes stretches of 11 consecutive negatively charged amino acids and 12 consecutive histidines. The sequence of the 8 protein was found to be essentially identical to a concurrently cloned human transcription factor that acts both positively and negatively in the context of immunoglobulin enhancers and a viral promoter. Our structural modeling of this protein indicates properties that could endow it with exquisite functional versatility.that encodes a protein termed 6, which binds to critical downstream elements in the genes encoding mouse ribosomal proteins L30 and L32 (rpL30 and rpL32) (Fig. 1A) and to downstream elements of several other genes, as inferred from sequence comparisons of functionally important factor binding sites (Fig. 1B) and a direct competition assay (10).tAt the completion of this study, we learned that the 6 sequence is almost identical to that of a human transcription factor that can act either negatively or positively in the context of the immunoglobulin K 3' and heavy-chain enhancers (13) and the P5 promoter of adeno-associated virus (14). The properties of this protein apparently suit it to function with downstream promoter elements, as well as to perform both activating and repressive functions. Our results have revealed that 8 is a zinc finger protein with unusual structural features that could endow it with exquisite functional versatility.The transcription of eukaryotic genes by RNA polymerase II is regulated by an interplay of sequence-specific activator or repressor factors and essentially nonspecific general factors and chromatin components (1). The specific factors recognize sequence modules or elements that are situated both in promoters-i.e., regions near the transcriptional start siteand in enhancers-i.e., regions distant from the start site. Specificity in transcriptional regulation is determined by the composition and organization of these elements and the availability of the factors that interact with them.A considerable effort is currently being made to understand the way in which sequence-specific and general factors interact during formation of active transcription complexes (2, 3). In most mechanistic studies of promoter function, major attention has been given to factors that bind to elements located upstream of the transcriptional start site. However, it has become increasingly evident that a large number of both cellular and viral genes utilize elements that are located downst...

An analysis of the methylated constituents of L cell mRNA by a combination of chromatographic methods and enzymatic treatments indicates that they comprise both 2'-O-methyl nucleosides and N6-methyl adenine, and/or 1-methyl adenine, and suggests that the 2'-O-methyl nucleotides, Ym, are part of an unusual class of sequences forming the 5' terminus of mRNA. These sequences seem to contain two 2'-O-methyl residues and a terminal residue that is not phosphorylated but, nevertheless, is blocked with respect to polynucleotid kinase reactivity. A strong candidate is a sequence of the type XppY1mpY2mpZp..., where X represents a blocking group which is itself occasionally methylated. The sequences isolated from total poly(A)+ mRNA contain all four species of 2'-O-methylated nucleoside, indicating some variability among different mRNA species. The methylated sequences do not appear to be enriched in the mRNA which hybridizes with repetitive DNA. The average L cell mRNA molecule also contains three residues of N6-methyl adenine. These residues are not part of the poly(A) segment, but appear to be located internal to the poly(A) near the 3' end of the mRNA molecules.

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The promoters of the mouse ribosomal protein genes rpL30, rpL32, and rpS16 are of equal strength, as indicated by in vivo measurements of polymerase loading and by their relative efficiency in driving the expression of a linked reporter gene. The equipotency of these promoters appears to derive from a remarkably similar architecture in which five or more elements are distributed over a 200-bp region that spans a polypyrimidineembedded cap site. Three trans-acting factors are shared by the rpL30 and rpL32 promoters, one of which, 8, recognizes a common CNGCCATCT motif in the first (untranslated) exons. Site-specific mutagenesis demonstrated that 8-factor binding is critical for rpL30 promoter function. The repeated occurrence of this novel promoter architecture among ribosomal protein genes with very different coding specificities is most readily explained by convergent evolution.

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