The KH domain mediates RNA binding in a wide range of proteins. Here we investigate the RNA-binding properties of two abundant RNA-binding proteins, ␣CP-2KL and heterogeneous nuclear ribonucleoprotein (hnRNP) K. These proteins constitute the major poly(C) binding activity in mammalian cells, are closely related on the basis of the structures and positioning of their respective triplicated KH domains, and have been implicated in a variety of post-transcriptional controls. By using SELEX, we have obtained sets of high affinity RNA targets for both proteins. The primary and secondary structures necessary for optimal protein binding were inferred in each case from SELEX RNA sequence comparisons and confirmed by mutagenesis and structural mapping. The target sites for ␣CP-2KL and hnRNP K were both enriched for cytosine bases and were presented in a single-stranded conformation. In contrast to these shared characteristics, the optimal target sequence for hnRNP K is composed of a single short "Cpatch" compatible with recognition by a single KH domain whereas that for ␣CP-2KL encompassed three such C-patches suggesting more extensive interactions. The binding specificities of the respective SELEX RNAs were confirmed by testing their interactions with native proteins in cell extracts, and the importance of the secondary structure in establishing an optimized ␣CP-2KL-binding site was supported by comparison of SELEX target structure with that of the native human ␣-globin 3-untranslated region. These data indicate that modes of macromolecular interactions of arrayed KH domains can differ even among closely related KH proteins and that binding affinities are substantially dependent on the presentation of the target site within the RNA secondary structure.Post-transcriptional controls play an important role in the determination of gene expression. The controls over RNA splicing, transport, localization, translation, and/or stability either contribute to or are the major component(s) of gene modulation during development (1). These controls are often mediated via interactions between specific mRNA sequences and/or structures and corresponding trans-acting RNA-binding proteins (2, 3). In several cases such interactions have been described in detail and emphasize the importance of primary and higher order structural RNA motifs (4 -9). The number and variety of RNA-binding proteins reported in the literature are rapidly expanding. Some of these RNA-binding proteins, such as those associated with heterogenous nuclear RNA, show low sequence specificity, suggesting general packaging functions (3, 10). Others demonstrate high level RNA-binding specificity suggesting circumscribed functions in gene control. Examples of the latter group of proteins include cytosolic iron-response element-binding protein (6, 11), human immunodeficiency virus Rev response element-binding protein (8), and the sex-lethal alternative-splicing factor (13). RNA-binding proteins, like their DNA-binding protein counterparts, tend to be modular in structure wit...

Globin mRNAs accumulate to 95% of total cellular mRNA during terminal erythroid differentiation, reflecting their extraordinary stability. The stability of human alpha-globin mRNA is paralleled by formation of a sequence-specific RNA-protein (RNP) complex at a pyrimidine-rich site within its 3' untranslated region (3'UTR), the alpha-complex. The proteins of the alpha-complex are widely expressed. The alpha-complex or a closely related complex also assembles at pyrimidine-rich 3'UTR segments of other stable mRNAs. These data suggest that the alpha-complex may constitute a general determinant of mRNA stability. One or more alphaCPs, members of a family of hnRNP K-homology domain poly(C) binding proteins, are essential constituents of the alpha-complex. The ability of alphaCPs to homodimerize and their reported association with additional RNA binding proteins such as AU-rich binding factor 1 (AUF1) and hnRNP K have suggested that the alpha-complex is a multisubunit structure. In the present study, we have addressed the composition of the alpha-complex. An RNA titration recruitment assay revealed that alphaCPs were quantitatively incorporated into the alpha-complex in the absence of associated AUF1 and hnRNP K. A high-affinity direct interaction between each of the three major alphaCP isoforms and the alpha-globin 3'UTR was detected, suggesting that each of these proteins might be sufficient for alpha-complex assembly. This sufficiency was further supported by the sequence-specific binding of recombinant alphaCPs to a spectrum of RNA targets. Finally, density sedimentation analysis demonstrated that the alpha-complex could accommodate only a single alphaCP. These data established that a single alphaCP molecule binds directly to the alpha-globin 3'UTR, resulting in a simple binary structure for the alpha-complex.

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We have characterized an unusual type of termination signal for T7 RNA polymerase that requires a conserved 7-base pair sequence in the DNA (ATCTGTT in the nontemplate strand). Each of the nucleotides within this sequence is critical for function, as any substitutions abolish termination. The primary site of termination occurs 7 nucleotides downstream from this sequence but is context-independent (that is, the sequence around the site of termination, and in particular the nucleotide at the site of termination, need not be conserved). Termination requires the presence of the conserved sequence and its complement in duplex DNA and is abolished or diminished if the signal is placed downstream of regions in which the non-template strand is missing or mismatched. Under the latter conditions, much of the RNA product remains associated with the template. The latter results suggest that proper resolution of the transcription bubble at its trailing edge and/or displacement of the RNA product are required for termination at this class of signal.A variety of signals have been found to modulate the process of transcript elongation. In general, these have been categorized as falling into the following three classes: pause sites, which temporarily halt the RNA polymerase (RNAP) 1 but subsequently allow resumption of transcription; termination signals, which cause release of the RNA and dissociation of the transcription complex; and arrest sites, at which the RNAP may be halted for a prolonged period but may escape by cleavage and subsequent elongation of the transcript (for review, see Refs. 1 and 2). Among the termination signals, the best characterized involve the formation of a stem-loop structure in the nascent RNA (3-5). Although there have been reports of pause, arrest, or termination signals that do not involve the formation of a structured RNA (see for example Ref. 6), these signals have been less well studied. In this work, we have characterized a sequence-specific pause/termination signal for T7 RNAP and have identified the elements that are required for its function.Two types of signals are known to cause pausing and/or termination by T7 RNAP (7,8). Class I terminators, typified by the signal that is present in the late region of T7 DNA (T⌽), encode RNAs that have the potential to form stable stem-loop structures followed by a run of U residues. These features are reminiscent of many intrinsic terminators utilized by Escherichia coli RNA polymerase, and a number of bacterial termination signals have been shown to cause T7 RNAP to terminate (8 -13). Although the members of this class encode RNAs that share a typical secondary structure, they exhibit little sequence homology.A second type of termination signal recognized by T7 RNAP was first identified in the cloned human prepro-parathyroid hormone (PTH) gene (8,14). These signals (class II signals) do not encode RNAs with an apparent consistent secondary structure but share a common sequence (ATCTGTT, in the nontemplate strand (8,15,16); this work). Additional membe...

A mutant T7 RNA polymerase (T7 RNAP) having two amino-acid substitutions (Y639F and $641A) is altered in its specificity towards nucleotide substrates, but is not affected in the specificity of its interaction with promoter and terminator sequences. The mutant enzyme gains the ability to utilize dNTPs and catalyze RNA and DNA synthesis from circular supercoiled plasmid DNA. DNA synthesis can also be initiated from a single stranded template using a DNA primer. Another T7 RNAP mutant having only the single substitution $641A loses RNA polymerase activity but is able to synthesize DNA.Key words: T7 RNA polymerase; Mutagenesis; dNTP utilization; DNA polymerizing activity distribution of hydroxyl-containing amino-acid residues. Specifically, a serine residue is present at position 641 in T7 RNAP (and at corresponding positions in related RNAPs), while in DNA polymerases (DNAP) no such regularity is observed [5]. As $641 is the hydroxyl-bearing amino-acid residue closest to Y639 we have asked whether the hydroxyl groups of these two residues may be involved in the interactions of enzyme with NTP and, specifically, in discrimination between dNTP and rNTP as potential substrates. To test this, we have generated mutant enzymes with phenylalanine in place of tyrosine at position 639, alanine in place of serine in position 641 and a double mutant bearing both of these substitutions. The substrate specificity and other features of the latter two proteins were found to be quite surprising.

A highly selective affinity labeling of T7 RNA polymerase with the o-formylphenyl ester of GMP and [a-