Interaction of the C-terminal domain of the E. coli RNA polymerase alpha subunit with the UP element: recognizing the backbone structure in the minor groove surface.

The C-terminal domain of the alpha-subunit of Escherichia coli RNA polymerase (alphaCTD) is responsible for transcriptional activation through interaction with both activator proteins and UP element DNA. Previously, we determined the solution structure of alphaCTD. Here, we investigated the interaction between alphaCTD and UP element DNA by NMR. DNA titration curves and intermolecular NOE measurements indicate that alphaCTD can bind to multiple sites on the UP element DNA. Unlike many transcription factors, alphaCTD does not have a strict base sequence requirement for binding. There is a good correlation between the strength of the interaction and the extent of intrinsic bending of the DNA oligomer estimated from the gel retardation assay. We propose that alphaCTD recognizes the backbone structure of DNA oligomers responsible for the intrinsic bending. Moreover, NMR studies and drug competition experiments indicated that alphaCTD interacts with the UP element on the minor groove side of the DNA. The C-terminal end of helix-1, the N-terminal end of helix-4, and the loop between helices 3 and 4 are used for the interaction. Based on these observations, we propose a model for the UP element-alphaCTD complex.

Structural study of DNA duplexes containing the (6-4) photoproduct by fluorescence resonance energy transfer.

Fluorescence resonance energy transfer (FRET) experiments have been performed to elucidate the structural features of oligonucleotide duplexes containing the pyrimidine(6-4)pyrimidone photoproduct, which is one of the major DNA lesions formed at dipyrimidine sites by UV light. Synthetic 32mer duplexes with and without the (6-4) photoproduct were prepared and fluorescein and tetramethylrhodamine were attached, as a donor and an acceptor, respectively, to the aminohexyl linker at the C5 position of thymine in each strand. Steady-state and time-resolved analyses revealed that both the FRET efficiency and the fluorescence lifetime of the duplex containing the (6-4) photoproduct were almost identical to those of the undamaged duplex, while marked differences were observed for a cisplatin-modified duplex, as a model of kinked DNA. Lifetime measurements of a series of duplexes containing the (6-4) photoproduct, in which the fluorescein position was changed systematically, revealed a small unwinding at the damage site, but did not suggest a kinked structure. These results indicate that formation of the (6-4) photoproduct induces only a small change in the DNA structure, in contrast to the large kink at the (6-4) photoproduct site reported in an NMR study.

Solution structure of an RNA duplex including a C-U base pair.

The formation of the C-U base pair in a duplex was observed in solution by means of the temperature profile of (15)N chemical shifts, and the precise geometry of the C-U base pair was also determined by NOE-based structure calculation. From the solution structure of the RNA oligomer, r[CGACUCAGG].r[CCUGCGUCG], it was found that a single C-U mismatch preferred being stacked in the duplex rather than being flipped-out even in solution. Moreover, it adopts an irregular geometry, where the amino nitrogen (N4) of the cytidine and keto-oxygen (O4) of the uridine are within hydrogen-bonding distance, as seen in crystals. To further prove the presence of a hydrogen bond in the C-U pair, we employed a point-labeled cytidine at the exocyclic amino nitrogen of the cytidine in the C-U pair. The temperature profile of its (15)N chemical shift showed a sigmoidal transition curve, indicating the presence of a hydrogen bond in the C-U pair in the duplex.

Regions essential for the interaction between Bcl-2 and SMN, the spinal muscular atrophy disease gene product.

The SMN gene is implicated in spinal muscular atrophy (SMA), and its product has been shown to interact with Bcl-2 protein to enhance its anti-apoptotic activity. In this study, we determined the regions that were essential for the interaction of Bcl-2 and SMN by co-immunoprecipitation of deletion mutants. Bcl-2 lacking its amino-terminal 20 amino acid residues or its carboxyl-terminal membrane-anchoring domain showed no or greatly reduced binding with SMN, respectively. However, Bcl-2 lacking other regions could still bind to SMN. Because Bcl-2 lacking the membrane-anchoring domain could bind to SMN in a yeast two-hybrid system, the amino-terminal region of Bcl-2 seems to be the most important domain for binding with SMN. A fragment of SMN encoded by exon 6 could bind to Bcl-2, but SMN lacking this region could not. From these results, we concluded that Bcl-2 and SMN proteins bound with each other at the amino-terminal region near the BH4 domain of Bcl-2 and the region encoded by exon 6 of SMN, both regions known to be important for their function.

Translational induction of heat shock transcription factor sigma32: evidence for a built-in RNA thermosensor.

Induction of heat shock proteins in Escherichia coli is primarily caused by increased cellular levels of the heat shock sigma-factor sigma32 encoded by the rpoH gene. Increased sigma32 levels result from both enhanced synthesis and stabilization. Previous work indicated that sigma32 synthesis is induced at the translational level and is mediated by the mRNA secondary structure formed within the 5'-coding sequence of rpoH, including the translation initiation region. To understand the mechanism of heat induction of sigma32 synthesis further, we analyzed expression of rpoH-lacZ gene fusions with altered stability of mRNA structure before and after heat shock. A clear correlation was found between the stability and expression or the extent of heat induction. Temperature-melting profiles of mRNAs with or without mutations correlated well with the expression patterns of fusion genes carrying the corresponding mutations in vivo. Furthermore, temperature dependence of mRNA-30S ribosome-tRNAfMet complex formation with wild-type or mutant mRNAs in vitro agreed well with that of the expression of gene fusions in vivo. Our results support a novel mechanism in which partial melting of mRNA secondary structure at high temperature enhances ribosome entry and translational initiation without involvement of other cellular components, that is, intrinsic mRNA stability controls synthesis of a transcriptional regulator.