The status of the Archaea as one of the three primary Domains emphasizes the importance of understanding their molecular fundamentals. Basic transcription in the Archaea resembles eucaryal transcription. However, little is known about transcriptional regulation. We have taken an in vivo approach, using genetics to address transcriptional regulation in the methanogenic Archaeon Methanococcus maripaludis. We identified a repressor binding site that regulates nif (nitrogen fixation) gene expression. The repressor binding site was palindromic (an inverted repeat) and was located just after the transcription start site of nifH. Mutations that changed the sequence of the palindrome resulted in marked decreases in repression by ammonia, even when the palindromic nature of the site was retained. The same mutations greatly decreased binding to the site by components of cell extract. These results provide the first partial description of a transcriptional regulatory mechanism in the methanogenic Archaea. This work also illustrates the utility of genetic approaches in Methanococcus that have not been widely used in the methanogens: directed mutagenesis and reporter gene fusions with lacZ.
Deficiency in the IKAP/Elp1 protein leads to the recessive sensory autosomal congenital neuropathy which is called familial dysautonomia (FD). This protein was originally identified as a role player in transcriptional elongation being a subunit of the RNAPII transcriptional Elongator multi-protein complex. Subsequently, IKAP/Elp1 was shown to play various functions in the cytoplasm. Here, we describe experiments performed with IKAP/Elp1 downregulated cell lines and FD-derived cells and tissues. Immunostaining of the cytoskeleton component α-tubulin in IKAP/Elp1 downregulated cells revealed disorganization of the microtubules (MTs) that was reflected in aberrant cell shape and process formation. In contrast to a recent report on the decrease in α-tubulin acetylation in IKAP/Elp1 downregulated cells, we were unable to observe any effect of IKAP/Elp1 deficiency on α-tubulin acetylation in the FD cerebrum and in a variety of IKAP/Elp1 downregulated cell lines. To explore possible candidates involved in the observed aberrations in MTs, we focused on superior cervical ganglion-10 protein (SCG10), also called STMN2, which is known to be an MT destabilizing protein. We have found that SCG10 is upregulated in the IKAP/Elp1-deficient FD cerebrum, FD fibroblasts and in IKAP/Elp1 downregulated neuroblastoma cell line. To better understand the effect of IKAP/Elp1 deficiency on SCG10 expression, we investigated the possible involvement of RE-1-silencing transcription factor (REST), a known repressor of the SCG10 gene. Indeed, REST was downregulated in the IKAP/Elp1-deficient FD cerebrum and IKAP/Elp1 downregulated neuroblastoma cell line. These results could shed light on a possible link between IKAP/Elp1 deficiency and cytoskeleton destabilization.
The glnA gene in the domains Bacteria andArchaea encodes glutamine synthetase, a universally distributed enzyme that functions in ammonia assimilation and glutamine synthesis. We investigated the regulation and function ofglnA in the methanogenic archaeon Methanococcus maripaludis. The deduced amino acid sequence of the gene demonstrated its membership in class GSI-α of glutamine synthetases. The gene appeared to be expressed as a monocistronic operon.glnA mRNA levels and specific activities of glutamine synthetase were regulated similarly by nitrogen. Three transcription start sites were identified, corresponding to two overlapping nitrogen-regulated promoters and one weaker constitutive promoter. An inverted repeat immediately upstream of the regulated transcription start sites mediated repression under noninducing conditions. Thus, mutations that altered the sequence of the inverted repeat resulted in derepression. The inverted repeat had sequence similarity with a repeat that we previously identified as the nif operator ofM. maripaludis, suggesting a common mechanism of nitrogen regulation. Efforts to produce a glnA null mutant failed, suggesting that glnA is an essential gene in M. maripaludis.
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