Many transcription factors, including the Escherichia coli cyclic AMP receptor protein (CRP), act by making direct contacts with RNA polymerase. At Class II CRP-dependent promoters, CRP activates transcription by making two such contacts: (i) an interaction with the RNA polymerase alpha subunit C-terminal domain (alphaCTD) that facilitates initial binding of RNA polymerase to promoter DNA; and (ii) an interaction with the RNA polymerase alpha subunit N-terminal domain that facilitates subsequent promoter opening. We have used random mutagenesis and alanine scanning to identify determinants within alphaCTD for transcription activation at a Class II CRP-dependent promoter. Our results indicate that Class II CRP-dependent transcription requires the side chains of residues 265, 271, 285-288 and 317. Residues 285-288 and 317 comprise a discrete 20x10 A surface on alphaCTD, and substitutions within this determinant reduce or eliminate cooperative interactions between alpha subunits and CRP, but do not affect DNA binding by alpha subunits. We propose that, in the ternary complex of RNA polymerase, CRP and a Class II CRP-dependent promoter, this determinant in alphaCTD interacts directly with CRP, and is distinct from and on the opposite face to the proposed determinant for alphaCTD-CRP interaction in Class I CRP-dependent transcription.

The opening of duplex DNA in the elongation phase of transcription by Escherichia coli RNA polymerase in vivo was detected at a regulatory site where a prolonged pause in transcription occurs. Single-stranded DNA in the transcription bubble was identified by its reactivity with potassium permanganate (KMnO4). The elongation structure in vivo was similar to that of transcription complexes made in vitro with some differences. The observed reactivity to KMnO4 of the DNA template strand was consistent with the existence of an RNA-DNA hybrid of about 12 nucleotides.

TK mRNA levels were determined in mouse L cells transformed with intron deletion mutations of the chicken TK gene. Whether normalized per cell, per integrated gene, or per internal control signal, intron deletion did not diminish the efficiency of TK mRNA formation in transformed L cells. The results demonstrated that introns are not required for efficient biogenesis of cellular mRNA in transformed mouse L cells.The general importance of introns for efficient gene expression in mammalian cells is an unresolved issue. Early work with recombinant simian virus 40 showed convincingly that efficient formation of viral 165 mRNA requires the presence of an intron in the DNA template (10,12,13,15,16); the intron requirement was manifested at a posttranscriptional level and could be satisfied by substituting an intron from a heterologous gene. These results suggested that passage through a splicing pathway might be a general requirement for formation of stable cytoplasmic mRNA. Such a requirement could explain the poor transformation efficiency of various intronless minigenes (5,16,17). However, rigorous reaffirmation of the importance of introns to eucaryotic mRNA formation has not been reported. In fact, for certain viral, plant, and yeast genes, evidence to the contrary has accumulated. Wild-type and intronless derivatives of the genes encoding adenovirus ElA protein (2, 25), polyomavirus T antigens (26,27), and the Rous sarcoma virus envelope protein (3) were equally efficient in generating mRNA in infected cells. Similar results were obtained for bean phaseolin in transformed plants (4) and Saccharomyces cerevisiae actin in transformed S. cerevisiae (21). Given these exceptions, a careful investigation of the importance of introns to expression of cellular genes in mammalian cells was warranted.Direct comparison of mRNA levels in mammalian cells transformed with wild-type and intronless cellular genes has not been reported. Hofbauer et al. (14) To investigate whether introns were required for efficient expression of cellular genes in animal cells, a series of intron deletion mutations of the chicken TK gene were constructed and transformed into L cells, and their level of expression was quantitated. The full-length chicken TK gene is interrupted by six introns. A seventh intron, in the 3' nontranslated region, is removed from rare TK mRNAs in some tissues (20). Intron deletion mutations of the chicken TK gene were made by combining cDNA and genomic fragments at shared restriction sites (Fig. 1). The mutations were named for the introns that were deleted from the gene. For example, the mutation Ail-2 lacks introns 1 and 2. Except for the removal of introns, all mutants were otherwise native and used the normal TK promoter and polyadenylation signals.As an initial test of the effect of intron deletion on gene expression, the mutants shown in Fig. 1 were used to transform TK-L cells to a HAT-resistant phenotype. The transformation efficiency of the different mutations relative to that of the full-length gene was deter...

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A segment ofEscherichia coli bacteriophage 21 DNA encoding the late-gene regulator, Q21, and the late-gene leader RNA segment was sequenced; its structure is similar to those of the related phages K and 82. The leader RNA is about 45 nudeotides long and consists essentiafly entirely of sequences encoding the p-independent terminator that is the putative target of the antitermination activity of Q21. Like the corresponding regions of A and 82, the 21 late-gene promoter segment encodes an early transcription pause in vitro, at about nucleotide 18, during which Q21 presumably acts to modify RNA polymerase. The 21 Q gene, cloned in isolation, is active on the late-gene leader segment in arns, and its purified product is active as an antiterminator in vitro; Q21 represents a third late-gene antiterminator, in addition to those of K and 82. There is little evident similarity in the primary sequences of the three Q genes.Growth of the lambdoid phages is regulated at the level of transcription termination (for a review, see references 4 and 17). The transcription antiterminators encoded by bacteriophage A genes N and Q (5) activate expression of the phage early and late genes, respectively, by allowing Escherichia coli RNA polymerase to transcribe through terminators located upstream of regulated genes. Phage X Q protein (QX) activates phage late-gene expression by allowing elongation of the constitutively expressed 6S leader RNA, which starts at the phage late-gene promoter (PR') and stops in the absence of QX at a strong p-independent terminator (tR') that precedes the late genes (9). The nucleic acid sequences required for QX function (qutX, for QX utilization) span the RNA start site and include the promoter elements and part of the adjacent transcribed region (25, 28). The late-gene regulatory region of phage 82 has a similar structure and mechanism of action (6); it also encodes a late leader RNA, named 82a. Other lambdoid phages, such as 21, 480, and the Salmonella typhimurium phage P22, also encode small RNA transcripts like X 6S that arise from corresponding regions of their genomes and that are putative leader sequences for late-gene expression (21).Q proteins are genome specific in function, despite their presumably common mechanism of action. QX acts on the phage X late-gene promoter or the nearly identical phage P22 late-gene promoter (8, 18) but not on the phage 82 late-gene promoter. Similarly, phage 82 Q protein (Q82) acts on the phage 82 genome but not on the phage X genome (6, 27), although it may share specificity with phage +80 Q protein (22). Therefore, Q proteins presumably interact with at least two transcription components: with DNA or RNA, to account for the genome specificity, and with RNA polymerase, to account for their action as antiterminators at various sites distant from the recognition and interaction site.The details of the molecular interactions between the Q proteins and these specific sequences are unknown. However, part of the mechanism of Q protein function is known and furthermore...