RESUMO
Protein translation can be affected by changes in the secondary structure of mRNA. The dinQ gene in Escherichia coli encodes a primary transcript (+1) that is inert to translation. Ribonucleolytic removal of the 44 first nucleotides converts the +1 transcript into a translationally active form, but the mechanism behind this structural change is unknown. Here we present experimental evidence for a mechanism where alternative RNA secondary structures in the two dinQ mRNA variants affect translation initiation by mediating opening or closing of the ribosome binding sequence. This structural switch is determined by alternative interactions of four sequence elements within the dinQ mRNA and also by the agrB antisense RNA. Additionally, the structural conformation of +1 dinQ suggests a locking mechanism comprised of an RNA stem that both stabilizes and prevents translation initiation from the full-length dinQ transcript. BLAST search and multiple sequence alignments define a new family of dinQ-like genes widespread in Enterobacteriaceae with close RNA sequence similarities in their 5' untranslated regions. Thus, it appears that a whole new family of genes is regulated by the same mechanism of alternative secondary RNA structures.
Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Proteínas de Membrana/genética , Conformação de Ácido Nucleico , Biossíntese de Proteínas , Regiões 5' não Traduzidas , Escherichia coli/metabolismo , Estabilidade de RNA , RNA Bacteriano/química , RNA Bacteriano/metabolismo , Alinhamento de SequênciaRESUMO
The functions of several SOS regulated genes in Escherichia coli are still unknown, including dinQ. In this work we characterize dinQ and two small RNAs, agrA and agrB, with antisense complementarity to dinQ. Northern analysis revealed five dinQ transcripts, but only one transcript (+44) is actively translated. The +44 dinQ transcript translates into a toxic single transmembrane peptide localized in the inner membrane. AgrB regulates dinQ RNA by RNA interference to counteract DinQ toxicity. Thus the dinQ-agr locus shows the classical features of a type I TA system and has many similarities to the tisB-istR locus. DinQ overexpression depolarizes the cell membrane and decreases the intracellular ATP concentration, demonstrating that DinQ can modulate membrane-dependent processes. Augmented DinQ strongly inhibits marker transfer by Hfr conjugation, indicating a role in recombination. Furthermore, DinQ affects transformation of nucleoid morphology in response to UV damage. We hypothesize that DinQ is a transmembrane peptide that modulates membrane-dependent activities such as nucleoid compaction and recombination.
Assuntos
Membrana Celular , Proteínas de Escherichia coli/genética , Escherichia coli , Proteínas de Membrana/genética , RNA Bacteriano , Membrana Celular/genética , Membrana Celular/metabolismo , Membrana Celular/efeitos da radiação , Citoplasma , Dano ao DNA/efeitos da radiação , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos da radiação , Peptídeos/genética , Peptídeos/metabolismo , RNA Antissenso/genética , RNA Antissenso/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Recombinação Genética/genética , Resposta SOS em Genética/efeitos da radiação , Transativadores/genética , Transativadores/metabolismo , Raios UltravioletaRESUMO
The DNA damage induced SOS response in Escherichia coli is initiated by cleavage of the LexA repressor through activation of RecA. Here we demonstrate that overexpression of the SOS-inducible tisAB gene inhibits several SOS functions in vivo. Wild-type E. coli overexpressing tisAB showed the same UV sensitivity as a lexA mutant carrying a noncleavable version of the LexA protein unable to induce the SOS response. Immunoblotting confirmed that tisAB overexpression leads to higher levels of LexA repressor and northern experiments demonstrated delayed and reduced induction of recA mRNA. In addition, induction of prophage lambda and UV-induced filamentation was inhibited by tisAB overexpression. The tisAB gene contains antisense sequences to the SOS-inducible dinD gene (16 nt) and the uxaA gene (20 nt), the latter encoding a dehydratase essential for galacturonate catabolism. Cleavage of uxaA mRNA at the antisense sequence was dependent on tisAB RNA expression. We showed that overexpression of tisAB is less able to confer UV sensitivity to the uxaA dinD double mutant as compared to wild-type, indicating that the dinD and uxaA transcripts modulate the anti-SOS response of tisAB. These data shed new light on the complexity of SOS regulation in which the uxaA gene could link sugar metabolism to the SOS response via antisense regulation of the tisAB gene.
Assuntos
Proteínas de Bactérias/biossíntese , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , RNA Mensageiro/genética , Resposta SOS em Genética , Serina Endopeptidases/biossíntese , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Bacteriófago lambda/efeitos da radiação , Sequência de Bases , Escherichia coli/citologia , Escherichia coli/efeitos da radiação , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/metabolismo , Dados de Sequência Molecular , Mutagênese , Fases de Leitura Aberta , Plasmídeos/genética , Plasmídeos/isolamento & purificação , Prófagos/efeitos da radiação , RNA Antissenso/química , RNA Mensageiro/biossíntese , RNA Mensageiro/metabolismo , Recombinases Rec A/biossíntese , Recombinases Rec A/genética , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Serina Endopeptidases/genética , Raios UltravioletaRESUMO
Several methods exist for predicting non-coding RNA (ncRNA) genes in Escherichia coli (E.coli). In addition to about sixty known ncRNA genes excluding tRNAs and rRNAs, various methods have predicted more than thousand ncRNA genes, but only 95 of these candidates were confirmed by more than one study. Here, we introduce a new method that uses automatic discovery of sequence patterns to predict ncRNA genes. The method predicts 135 novel candidates. In addition, the method predicts 152 genes that overlap with predictions in the literature. We test sixteen predictions experimentally, and show that twelve of these are actual ncRNA transcripts. Six of the twelve verified candidates were novel predictions. The relatively high confirmation rate indicates that many of the untested novel predictions are also ncRNAs, and we therefore speculate that E.coli contains more ncRNA genes than previously estimated.
Assuntos
Biologia Computacional/métodos , Escherichia coli/genética , Genes Bacterianos , RNA não Traduzido/genética , Análise de Sequência de DNA/métodos , Algoritmos , Genes de RNAr , RNA de Transferência/genética , RNA não Traduzido/análiseRESUMO
Enzymes involved in genomic maintenance of human parasites are attractive targets for parasite-specific drugs. The parasitic protozoan Trypanosoma cruzi contains at least two enzymes involved in the protection against potentially mutagenic uracil, a deoxyuridine triphosphate nucleotidohydrolase (dUTPase) and a uracil-DNA glycosylase belonging to the highly conserved UNG-family. Uracil-DNA glycosylase activities excise uracil from DNA and initiate a multistep base-excision repair (BER) pathway to restore the correct nucleotide sequence. Here we report the biochemical characterisation of T.cruzi UNG (TcUNG) and its contribution to the total uracil repair activity in T.cruzi. TcUNG is shown to be the major uracil-DNA glycosylase in T.cruzi. The purified recombinant TcUNG exhibits substrate preference for removal of uracil in the order ssU>U:G>U:A, and has no associated thymine-DNA glycosylase activity. T.cruzi apparently repairs U:G DNA substrate exclusively via short-patch BER, but the DNA polymerase involved surprisingly displays a vertebrate POLdelta-like pattern of inhibition. Back-up UDG activities such as SMUG, TDG and MBD4 were not found, underlying the importance of the TcUNG enzyme in protection against uracil in DNA and as a potential target for drug therapy.
Assuntos
DNA Glicosilases/metabolismo , Reparo do DNA , Trypanosoma cruzi/enzimologia , Uracila/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Doença de Chagas/tratamento farmacológico , Doença de Chagas/parasitologia , DNA/química , DNA/genética , DNA/metabolismo , DNA Glicosilases/antagonistas & inibidores , DNA Glicosilases/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Teste de Complementação Genética , Humanos , Dados de Sequência Molecular , Mutação , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Trypanosoma cruzi/genética , Uracila-DNA GlicosidaseRESUMO
The alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) is known to trigger the adaptive response by inducing the ada-regulon - consisting of three DNA repair enzymes Ada, AlkB, AlkA and the enigmatic AidB. We have applied custom designed tiling arrays to study transcriptional changes in Escherichia coli following a MNNG challenge. Along with the expected upregulation of the adaptive response genes (ada, alkA and alkB), we identified a number of differentially expressed transcripts, both novel and annotated. This indicates a wider regulatory response than previously documented. There were 250 differentially-expressed and 2275 similarly-expressed unannotated transcripts. We found novel upregulation of several stress-induced transcripts, including the SOS inducible genes recN and tisAB, indicating a novel role for these genes in alkylation repair. Furthermore, the ada-regulon A and B boxes were found to be insufficient to explain the regulation of the adaptive response genes after MNNG exposure, suggesting that additional regulatory elements must be involved.
Assuntos
Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Metilnitronitrosoguanidina/farmacologia , Transcrição Gênica , Adaptação Biológica/genética , Perfilação da Expressão Gênica , Mutação , Óperon , Regiões Promotoras Genéticas , RNA não Traduzido/genética , Reprodutibilidade dos Testes , Transcriptoma , Regiões não TraduzidasRESUMO
BACKGROUND: Despite comprehensive investigation, the Escherichia coli SOS response system is not yet fully understood. We have applied custom designed whole genome tiling arrays to measure UV invoked transcriptional changes in E. coli. This study provides a more complete insight into the transcriptome and the UV irradiation response of this microorganism. RESULTS: We detected a number of novel differentially expressed transcripts in addition to the expected SOS response genes (such as sulA, recN, uvrA, lexA, umuC and umuD) in the UV treated cells. Several of the differentially expressed transcripts might play important roles in regulation of the cellular response to UV damage. We have predicted 23 novel small peptides from our set of detected non-gene transcripts. Further, three of the predicted peptides were cloned into protein expression vectors to test the biological activity. All three constructs expressed the predicted peptides, in which two of them were highly toxic to the cell. Additionally, a remarkably high overlap with previously in-silico predicted non-coding RNAs (ncRNAs) was detected. Generally we detected a far higher transcriptional activity than the annotation suggests, and these findings correspond with previous transcription mappings from E. coli and other organisms. CONCLUSIONS: Here we demonstrate that the E. coli transcriptome consists of far more transcripts than the present annotation suggests, of which many transcripts seem important to the bacterial stress response. Sequence alignment of promoter regions suggest novel regulatory consensus sequences for some of the upregulated genes. Finally, several of the novel transcripts identified in this study encode putative small peptides, which are biologically active.
Assuntos
Escherichia coli/metabolismo , Regulação da Expressão Gênica , Peptídeos/genética , Sequência de Bases , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Fases de Leitura Aberta , Peptídeos/química , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , RNA não Traduzido/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Resposta SOS em Genética , Transcrição Gênica , Raios UltravioletaRESUMO
Uracil may arise in DNA as a result of deamination of cytosine or through incorporation of dUMP instead of dTMP during replication. We have studied the steady-state levels of uracil in the DNA of primary cells and mouse embryonic fibroblast (MEF) cell lines from mice deficient in the Ung uracil-DNA glycosylase. The results show that the levels of uracil in the DNA of Ung(-/-) cells strongly depend on proliferation, indicating that the uracil residues originate predominantly from misincorporation during replication. Treatment with 5-fluoro-2'-deoxyuridine (5-FdUrd) or 5-fluorouracil (5-FU) gives rise to a dose-dependent increase of uracil in Ung(-/-) MEFs (up to 1.5-fold) but not in wild-type cells. Interestingly, Ung(-/-) MEFs accumulate AP-sites as well as uracil in response to 5-FdUrd but not to 5-FU. This accumulation of repair intermediates suggests a loss of tightly co-ordinated repair in the absence of Ung, and correlates with stronger inhibition of cell proliferation in response to 5-FdUrd, but not to 5-FU, in Ung(-/-) MEFs compared with wild-type cells. However, other cytotoxic effects of these fluoropyrimidines are comparable in both wild-type and Ung-deficient cells, demonstrating that excision of uracil from DNA by the Ung uracil-DNA glycosylase is not a prerequisite for obtaining cytotoxicity.