RESUMO
Expression of the Anabaena sp. strain PCC 7120 RNA helicase gene crhC is induced by cold shock. crhC transcripts are not detectable at 30 degrees C but accumulate at 20 degrees C, and levels remain elevated for the duration of the cold stress. Light-derived metabolic capability, and not light per se, is required for crhC transcript accumulation. Enhanced crhC mRNA stability contributes significantly to the accumulation of crhC transcripts, with the crhC half-life increasing sixfold at 20 degrees C. The accumulation is reversible, with the cells responding more rapidly to temperature downshifts than to upshifts, as a result of the lack of active mRNA destabilization and the continuation of crhC transcription, at least transiently, after a temperature upshift. Translational inhibitors do not induce crhC expression to cold shock levels, indicating that inhibition of translation is only one of the signals required to activate the cold shock response in Anabaena. Limited amounts of protein synthesis are required for the cold shock-induced accumulation of crhC transcripts, as normal levels of accumulation occur in the presence of tetracycline but are abolished by chloramphenicol. Regulation of crhC expression may also extend to the translational level, as CrhC protein levels do not correlate completely with the pattern of mRNA transcript accumulation. Our experiments indicate that the regulation of crhC transcript accumulation is tightly controlled by both temperature and metabolic activity at the levels of transcription, mRNA stabilization, and translation.
Assuntos
Anabaena/genética , Temperatura Baixa , RNA Helicases/genética , Anabaena/efeitos dos fármacos , Anabaena/enzimologia , Cloranfenicol/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , RNA/genética , RNA/metabolismo , RNA Helicases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Temperatura , Tetraciclina/farmacologia , Transcrição GênicaRESUMO
The ability to modify RNA secondary structure is crucial for numerous cellular processes. We have characterized two RNA helicase genes, crhB and crhC, which are differentially expressed in the cyanobacterium Anabaena sp. strain PCC 7120. crhC transcription is limited specifically to cold shock conditions while crhB is expressed under a variety of conditions, including enhanced expression in the cold. This implies that both RNA helicases are involved in the cold acclimation process in cyanobacteria; however, they presumably perform different roles in this adaptation. Although both CrhB and CrhC belong to the DEAD box subfamily of RNA helicases, CrhC encodes a novel RNA helicase, as the highly conserved SAT motif is modified to FAT. This alteration may affect CrhC function and its association with specific RNA targets and/or accessory proteins, interactions required for cold acclimation. Primer extension and analysis of the 5' untranslated region of crhC revealed the transcriptional start site, as well as a number of putative cold shock-responsive elements. The potential role(s) performed by RNA helicases in the acclimation of cyanobacteria to cold shock is discussed.
Assuntos
Anabaena/enzimologia , RNA Helicases/biossíntese , Sequência de Aminoácidos , Anabaena/genética , Sequência de Bases , Clonagem Molecular , Temperatura Baixa , Primers do DNA/genética , DNA Bacteriano/genética , Indução Enzimática , Expressão Gênica , Genes Bacterianos , Genes Reguladores , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Helicases/genética , Homologia de Sequência de AminoácidosRESUMO
Two Nicotiana plumbaginifolia cDNA clones, NeIF-5A1 and NeIF-5A2, encoding eukaryotic translation initiation factor eIF-5A (formerly called eIF-4D) were cloned by heterologous screening with Dictyostelium and human eIF-5A probes. eIF-5A is the only protein known to contain a unique amino acid modification, hypusine. Comparison of the Nicotiana deduced amino acid sequences with those of other eIF-5A polypeptides reveals conservation throughout the coding sequence, especially in the region of the hypusine residue. Transcript analysis reveals that NeIF-5A1 is preferentially expressed in photosynthetic tissues, while NeIF-5A2 is constitutively expressed in all plant tissues examined. A polyclonal antibody was raised against NeIF-5A1 overexpressed in E. coli. NeIF-5A1 antiserum crossreacts with an 18 kDa polypeptide doublet in all tobacco tissues examined. At least one polypeptide of ca. 18 kDa from a diversity of higher and lower plants crossreacts with NeIF-5A1 antiserum.
