Binding of transcription termination protein nun to nascent RNA and template DNA.

The amino-terminal arginine-rich motif of coliphage HK022 Nun binds phage lambda nascent transcript, whereas the carboxyl-terminal domain interacts with RNA polymerase (RNAP) and blocks transcription elongation. RNA binding is inhibited by zinc (Zn2+) and stimulated by Escherichia coli NusA. To study these interactions, the Nun carboxyl terminus was extended by a cysteine residue conjugated to a photochemical cross-linker. The carboxyl terminus contacted NusA and made Zn2+-dependent intramolecular contacts. When Nun was added to a paused transcription elongation complex, it cross-linked to the DNA template. Nun may arrest transcription by anchoring RNAP to DNA.

Thrombospondin-1 repression is mediated via distinct mechanisms in fibroblasts and epithelial cells.

Tumor-associated angiogenesis is postulated to be regulated by the balance between pro- and anti-angiogenic factors. We demonstrate here that the critical step in establishing the angiogenic capability of human tumor cells is the repression of a key secreted anti-angiogenic factor, thrombospondin-1 (Tsp-1). This repression is essential for tumor formation by mammary epithelial cells and kidney cells engineered to express SV40 early region proteins, hTERT, and H-RasV12. In transformed epithelial cells, a signaling pathway leading from Ras to Tsp-1 repression induces the sequential activation of PI3 kinase, Rho and ROCK, leading to activation of Myc through phosphorylation, thereby enabling Myc to repress Tsp-1 transcription. In transformed fibroblasts, however, the repression of Tsp-1 can be achieved by an alternative mechanism involving inactivation of both p53 and pRb. We thus describe novel mechanisms by which the activation of oncogenes in epithelial cells and the inactivation of tumor suppressors in fibroblasts permits angiogenesis and, in turn, tumor formation.

Escherichia coli NusA is required for efficient RNA binding by phage HK022 nun protein.

The Nun protein of phage HK022 is an RNA binding protein of the arginine-rich motif family. Nun binds the phage lambda boxB RNA sequence (BOXB) on nascent lambda transcripts and arrests transcription elongation. Binding to BOXB is inhibited by Zn2+ and stimulated by the Escherichia coli NusA protein. Deletion of the Nun C-terminal region enhances BOXB binding and makes it independent of Zn2+ and NusA. The C terminus of Nun thus appears to interfere with the N-terminal RNA binding motif. NusA relieves this interference by binding to the Nun C terminus and forming a complex with Nun and BOXB. However, NusA also inhibits transcription arrest in vitro, in the absence of the other Nus factors. Nun deleted for its C terminus fails to bind RNA polymerase (RNAP) (RNAP) or NusA in vitro or to arrest transcription in vivo or in vitro. Our findings are consistent with the idea that NusA inhibits transcription arrest by binding to the Nun C terminus, thus blocking the interaction between Nun and RNAP. NusG, NusB, and NusE factors restore transcription arrest, presumably by promoting transfer of Nun from NusA to RNAP.

The carboxyl terminus of phage HK022 Nun includes a novel zinc-binding motif and a tryptophan required for transcription termination.

The amino-terminal arginine-rich motif of the phage HK022 Nun protein binds phage lambda nascent mRNA transcripts while the carboxy-terminal domain binds RNA polymerase and arrests transcription. The role of specific residues in the carboxy-terminal domain in transcription termination were investigated by mutagenesis, in vitro and in vivo functional assays, and NMR spectroscopy. Coordination of zinc to three histidine residues in the carboxy-terminus inhibited RNA binding by the amino-terminal domain; however, only two of these histidines were required for transcription arrest. These results suggest that additional zinc-coordinating residues are supplied by RNA polymerase in the context of the Nun-RNA polymerase complex. Substitution of the penultimate carboxy-terminal tryptophan residue with alanine or leucine blocks transcription arrest, whereas a tyrosine substitution is innocuous. Wild-type Nun fails to arrest transcription on single-stranded templates. These results suggest that Nun inhibition of transcription elongation is due in part to interactions between the carboxy-terminal tryptophan of Nun and double-stranded DNA, possibly by intercalation. A model for the termination activity of Nun is developed on the basis of these data.