No abstract

The crystal structure of an engineered monomer of the lambda Cro repressor shows unexpected expansion of the hydrophobic core of the protein and disorder of the five C-terminal residues [Albright et al. (1996) Biochemistry 35, 735-742]. This structural information has guided the construction of a second generation of monomeric Cro proteins by combinatorial mutagenesis of selected core and C-terminal residues. Clones were identified in a library of randomized cro genes by a genetic screen for protein accumulation in Escherichia coli. Sequencing of candidate genes followed by purification and analysis of their product proteins has identified alternative arrangements of hydrophobic core residues which result in substantial increases in thermal stability. In contrast, residue replacements at the C-terminus have minor effects on stability but may increase protein expression levels.

High levels of RNA polymerase III gene transcription are achieved by facilitated recycling of the polymerase on transcription factor IIIB (TFIIIB)-DNA complexes that are stable through multiple rounds of initiation. TFIIIB-DNA complexes in yeast comprise the TATAbinding protein (TBP), the TFIIB-related factor TFIIIB70, and TFIIIB90. The high stability of the TFIIIB-DNA complex is conferred by TFIIIB90 binding to TFIIIB70-TBP-DNA complexes. This stability is thought to result from compound bends introduced in the DNA by TBP and TFIIIB90 and by protein-protein interactions that obstruct DNA dissociation. Here we present biochemical evidence that the high stability of TFIIIB-DNA complexes results from kinetic trapping of the DNA. Thermodynamic analysis shows that the free energies of formation of TFIIIB70-TBP-DNA (⌬G°‫؍‬ ؊12.10 ؎ 0.12 kcal͞mol) and TFIIIB-DNA (⌬G°‫؍‬ ؊11.90 ؎ 0.14 kcal͞mol) complexes are equivalent whereas a kinetic analysis shows that the half-lives of these complexes (46 ؎ 3 min and 95 ؎ 6 min, respectively) differ significantly. The differential stability of these isoenergetic complexes demonstrates that TFIIIB90 binding energy is used to drive conformational changes and increase the barrier to complex dissociation. RNA polymerase (pol) III transcribes a variety of nontranslated RNA genes encoding transfer RNAs, 5S ribosomal RNA, U6 snRNA, and other small cellular RNAs (1). In Saccharomyces cerevisiae, transcription of these genes is directed by the initiation factor TFIIIB, which is assembled upstream of the start site by other factors (TFIIIA and͞or TFIIIC), bound to downstream promoter elements. Yeast TFIIIB is a heterotrimeric complex comprising the TATA-binding protein (TBP), a TFIIB-related component, TFIIIB70 (Brf1), and a SANT domain protein, TFIIIB90 (BЈЈ). Structural and functional homologs of these proteins have been identified in human cells and confer TFIIIB activity (termed TFIIIB-␣ in ref.2) for the transcription of tRNA and related pol III genes having internal promoter elements (2-4). Additionally, human cells contain a second TFIIIB activity (TFIIIB-␤) that is used by pol III genes whose promoter elements are located upstream of the start site (e.g., U6 snRNA and 7SK RNA). TFIIIB-␤ differs from TFIIIB-␣ in that it contains a different TFIIB-related component (termed BRFU or hTFIIIB50) and associated proteins (2, 3). Further complexity among TFIIIB complexes is suggested by the identification of three splice variants of human Brf1 (4). One of these variants, Brf2, appears to be active in the transcription U6 snRNA.Pol III genes are among the most actively transcribed genes in eukaryotic cells. High rates of pol III gene transcription are achieved through the facilitated recycling of pol molecules (5-8) on TFIIIB complexes that remain bound to the DNA for multiple rounds of initiation (9,10). The stability of TFIIIB-DNA complexes is therefore a key property that enables rapid reinitiation by eliminating rate-limiting steps in transcription complex assembly. Yeast TFIIIB-DNA compl...