Charles J. Daniels

Research Description:

RNA Studies: RNA maturation studies have focused on the characterization of tRNA intron processing enzymes and the role of eucaryal small nucleolar RNA (snoRNA)-like RNAs in the processing and modification of archaeal stable RNAs. We have now established that the processing of intron-containing tRNAs in the Archaea is carried out by an enzyme system that is related to its eucaryal nuclear counterpart. This has led us to propose that archaeal tRNA and rRNA introns are related to the nuclear tRNA introns and that the two intron endonuclease enzyme systems evolved from a common origin. Although related at the level of protein sequence, these two enzymes have very different substrate recognition requirements. The mechanisms by which the eucaryal and archaeal tRNA intron endonucleses recognize their substrates remains an active question. We are currently examining the interaction of pretRNAs with the endonuclease in combination with mutagenesis studies to identify the catalytic domain of this unusual, structure-specific endoribonuclease. In parallel, we have recently initiated studies on the role of snoRNA-like RNAs and the nucleolar related proteins fibrillarin and Prp31 in the processing of stable RNA precursors in H. volcanii and Methanobacterium thermoautotrophicum. The Archaea appear to possess a RNA processing and modification system similar to that found in the nucleolus of eucaryal cells. These enzymes and their associated RNAs are likely to be involved rRNA and tRNA processing and modification in the Archaea.

Transcription Studies: We have developed an in vivo transcription assay for the halophilic archaeon, H. volcanii and utilized this system to identify sequences that are required for expression of tRNA and heat shock genes. Expression of the H. volcanii tRNALys gene requires two elements: the TATA-like element and a purine rich sequence located 5' of this sequence. Transcription start site selection for this gene is highly selective, initiating at a single site and showing a strong preference for a purine at the initiation site. Transcription termination in H. volcanii also appears to be complex. We have found that oligo-T sequences and bacterial rho-independent-like elements function as efficient terminators in this organism. As an example of regulated gene expression we have characterized the requirement for heat shock induced transcription of the H. volcanii Cct1 (TCP-1 of TF55-like) gene. Regulation of this gene requires the TATA-like element and sequences flanking this region. The pattern of these requirements generally supports the notion that transcription initiation and termination in the Archaea most closely resemble the eucaryal RNA polymerase II system. We have also begun an analysis of the transcription machinery of H. volcanii. Cloning and sequence analysis indicates that the H. volcanii, and the halophiles in general, possess the eucaryal-like transcription factors TATA binding protein (TBP) and TFIIB. Unexpectedly, we have found that H. volcanii has multiple TBP and TFIIB genes and that some of these genes are differently regulated. One such example is the TFIIB2 gene, which is specifically induced during heat shock.We are currently investigating the role(s) of these transcription factors in regulated gene expression to determine if these organisms use specific transcription factor pairing as a scheme to regulate gene expression.

Genome Analysis: In collaboration with Dr. J. N. Reeve, and Dr. Doug Smith of Genome Therapeutics, we are participants in the Department of Energy supported microbial genome sequence program. Sequence of the 1.7 Mb Methanobacterium thermoautotrophicum genome has been completed and we are continuing in the comparative analysis of this and other archaeal genomes. The gene content of this genome, as well as tools for searching the genome, are presented at our website .