Structural studies of the tRNA domain of tmRNA.

tmRNA is a small, stable prokaryotic RNA. It rescues ribosomes that have become stalled during the translation of mRNA fragments lacking stop codons, or during periods of tRNA scarcity. It derives its name from the presence of two separate domains, one that functions as a tRNA, and another that serves as an mRNA. We have carried out modeling and transient electric birefringence studies to determine the angle between the acceptor stem and anticodon stem of the tRNA domain of Eschericia coli tmRNA. The results of the modeling studies yielded an interstem angle of 110 degrees, in agreement with the lower end of the range of angles (111 degrees -137 degrees ) determined experimentally for various solution conditions. The range of experimental angles is greater than the angles observed for any of the tRNA crystal structures, in line with the presence of a shortened D stem. The secondary structure of the tRNA domain is conserved for all known tmRNA sequences, so we propose that the angle is also conserved. These results also suggest that the region of tmRNA between P2a and P2b may interact with the decoding site of the ribosome.

Determination of the angle between the acceptor and anticodon stems of a truncated mitochondrial tRNA.

Significant departures from the canonical (cloverleaf) secondary structure of transfer (t)RNAs can be found among the mitochondrial (m)tRNAs of higher metazoans; these mtRNAs thus pose a challenge to the concept of an invariant, L-shaped tertiary conformation for all tRNAs. For bovine mtRNASer(AGY), which lacks the entire "dihydrouridine" (dhU) arm, two distinct tertiary models have been proposed: the first model preserves the L-shaped conformation at the expense of overall size; the second model preserves the absolute distance between the 3' terminus and the anticodon loop, while allowing the acceptor-anticodon interstem angle to vary. We have tested the central predictions of these two models by performing a series of transient electric birefringence measurements on bovine mtRNASer(AGY) constructs in which the aminoacyl-acceptor and anticodon stems were each extended by approximately 70 bp. This mtRNA species is particularly amenable to analysis, since the native bovine (heart) mtRNA is completely unmodified outside of the anticodon loop. For magnesium ion concentrations above 1 mM, the interstem angle for the extended mtRNA, 120(+/-5) degrees, is approximately 50% larger than the corresponding angle for yeast tRNAPhe (70-80 degrees) under the same ionic conditions. Furthermore, the interstem angles of the two tRNAs exhibit strikingly different responses to the addition of magnesium ions: the interstem angle for yeast tRNAPhe is reduced by nearly 50 % upon addition of 2 mM magnesium ions, whereas the angle for mtRNASer(AGY) increases by about 10%. Our data thus support a central prediction of the second model; namely, that truncated mtRNAs will possess more open interstem angles. In addition, we demonstrate that birefringence amplitude data can be used to provide model-independent estimates for the interstem angles.

Cdc73p and Paf1p are found in a novel RNA polymerase II-containing complex distinct from the Srbp-containing holoenzyme.

The products of the yeast CDC73 and PAF1 genes were originally identified as RNA polymerase II-associated proteins. Paf1p is a nuclear protein important for cell growth and transcriptional regulation of a subset of yeast genes. In this study we demonstrate that the product of CDC73 is a nuclear protein that interacts directly with purified RNA polymerase II in vitro. Deletion of CDC73 confers a temperature-sensitive phenotype. Combination of the cdc73 mutation with the more severe paf1 mutation does not result in an enhanced phenotype, indicating that the two proteins may function in the same cellular processes. To determine the relationship between Cdc73p and Paf1p and the recently described holoenzyme form of RNA polymerase II, we created yeast strains containing glutathione S-transferase (GST)-tagged forms of CDC73, PAF1, and TFG2 functionally replacing the chromosomal copies of the genes. Isolation of GST-tagged Cdc73p and Paf1p complexes has revealed a unique form of RNA polymerase II that contains both Cdc73p and Paf1p but lacks the Srbps found in the holoenzyme. The Cdc73p-Paf1p-RNA polymerase II-containing complex also includes Gal11p, and the general initiation factors TFIIB and TFIIF, but lacks TBP, TFIIH, and transcription elongation factor TFIIS as well as the Srbps. The Srbp-containing holoenzyme does not include either Paf1p or Cdc73p, demonstrating that these two forms of RNA polymerase II are distinct. In confirmation of the hypothesis that the two forms coexist in yeast cells, we found that a TFIIF-containing complex isolated via the GST-tagged Tfg2p construct contains both (i) the Srbps and (ii) Cdc73p and Paf1p. The Srbps and Cdc73p-Paf1p therefore appear to define two complexes with partially redundant, essential functions in the yeast cell. Using the technique of differential display, we have identified several genes whose transcripts require Cdc73p and/or Paf1p for normal levels of expression. Our analysis suggests that there are multiple RNA polymerase II-containing complexes involved in the expression of different classes of protein-coding genes.

Functional components of fibroblast growth factor (FGF) signal transduction in pituitary cells. Identification of FGF response elements in the prolactin gene.

Fibroblast growth factors (FGFs) have been implicated in pituitary lactotroph tumorigenesis; however, little is known about the molecular mechanisms of FGF signal transduction. We used a transient transfection approach, in GH4 cells, to identify components of the FGF signaling pathway leading to activation of the rat prolactin (rPRL) promoter. Using dominant-negative constructs of p21(Ras), Raf-1 kinase, and mitogen-activated protein (MAP) kinase, we show that FGF activation of the rPRL promoter is independent of Ras and Raf-1 but requires MAP kinase. Furthermore, MAP kinase but not Raf-1 kinase catalytic activity is stimulated by FGFs. The rPRL promoter FGF response maps to two Ets binding sites, centered at -212 (FRE1) and -96 (FRE2), and co-transfection of dominant-negative Ets inhibits FGF activation. FRE1 co-localizes with a composite, Ets/GHF-1, Ras response element. However, overexpression of Ets-1 and GHF-1, which potentiate the Ras response, inhibits FGF stimulation of the rPRL promoter, implying that Ras and FGF signaling pathways target distinct factors to elicit their effects. These data suggest that Ets factors serve to sort and integrate MAP kinase-dependent growth factor signals, allowing highly specific transcriptional responses to be mediated via the interaction of distinct Ets proteins and cofactors at common response elements.