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1.
RNA ; 18(5): 1062-74, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22454537

RESUMO

Hfq-dependent sRNAs contain, at least, an mRNA base-pairing region, an Hfq-binding site, and a Rho-independent terminator. Recently, we found that the terminator poly(U) of Escherichia coli sRNAs is essential for Hfq binding and therefore for riboregulation. In this study, we tried to identify additional components within Hfq-binding sRNAs required for efficient Hfq binding by using SgrS as a model. We demonstrate by mutational and biochemical studies that an internal hairpin and an immediately upstream U-rich sequence also are required for efficient Hfq binding. We propose that the functional Hfq-binding module of SgrS consists of an internal hairpin preceded by a U-rich sequence and a Rho-independent terminator with a long poly(U) tail. We also show that the Rho-independent terminator alone can act as a functional Hfq-binding module when it is preceded by an internal U-rich sequence. The 3' region of most known sRNAs share the features corresponding to either a double- or single-hairpin-type Hfq-binding module. We also demonstrate that increasing the spacing between the base-pairing region and the Hfq-binding module reduces or impairs the silencing ability. These findings allowed us to design synthetic Hfq-binding sRNAs to target desired mRNAs.


Assuntos
Fator Proteico 1 do Hospedeiro/metabolismo , Sequências Repetidas Invertidas , Poli U/química , RNA Bacteriano/química , Composição de Bases , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Códon de Terminação , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , RNA Bacteriano/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo
2.
Proc Natl Acad Sci U S A ; 108(32): 13059-64, 2011 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-21788484

RESUMO

Major bacterial small RNAs (sRNAs) regulate the translation and stability of target mRNAs through base pairing with the help of the RNA chaperone Hfq. The Hfq-dependent sRNAs consist of three basic elements, mRNA base-pairing region, Hfq-binding site, and rho-independent terminator. Although the base-pairing region and the terminator are well documented in many sRNAs, the Hfq-binding site is less well-defined except that Hfq binds RNA with a preference for AU-rich sequences. Here, we performed mutational and biochemical studies to define the sRNA site required for Hfq action using SgrS as a model sRNA. We found that shortening terminator polyU tail eliminates the ability of SgrS to bind to Hfq and to silence ptsG mRNA. We also demonstrate that the SgrS terminator can be replaced with any foreign rho-independent terminators possessing a polyU tail longer than 8 without losing the ability to silence ptsG mRNA in an Hfq-dependent manner. Moreover, we found that shortening the terminator polyU tail of several other sRNAs also eliminates the ability to bind to Hfq and to regulate target mRNAs. We conclude that the polyU tail of sRNAs is essential for Hfq action in general. The data also indicate that the terminator polyU tail plays a role in Hfq-dependent stabilization of sRNAs.


Assuntos
Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/metabolismo , Fator Proteico 1 do Hospedeiro/metabolismo , Poli U/metabolismo , RNA Bacteriano/metabolismo , Regiões Terminadoras Genéticas/genética , Sequência Rica em At/genética , Sequência de Bases , Sítios de Ligação , Inativação Gênica , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Conformação de Ácido Nucleico , Ligação Proteica , RNA Bacteriano/química , RNA Bacteriano/genética
3.
Mol Microbiol ; 76(3): 782-92, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20345651

RESUMO

Escherichia coli SgrS is an Hfq-binding small RNA that is induced under glucose-phosphate stress to cause translational repression and RNase E-dependent rapid degradation of ptsG mRNA encoding the major glucose transporter. A 31-nt-long stretch in the 3' region of SgrS is partially complementary to the translation initiation region of ptsG mRNA. We showed previously that SgrS alone causes translational repression when pre-annealed with ptsG mRNA by a high-temperature treatment in vitro. Here, we studied translational repression of ptsG mRNA in vitro by synthetic RNA oligonucleotides (oligos) to define the SgrS region required for translational repression. We first demonstrate that a 31 nt RNA oligo corresponding to the base-pairing region is sufficient for translational inhibition of ptsG mRNA. Then, we show that RNA oligo can be shortened to 14 nt without losing its effect. Evidence shows that the 14 nt base-pairing region is sufficient to inhibit ptsG translation in the context of full-length SgrS in vivo. We conclude that SgrS 168-181 is a minimal base-pairing region for translational inhibition of ptsG mRNA. Interestingly, the 14 nt oligo efficiently inhibited ptsG translation without the high-temperature pre-treatment, suggesting that remodelling of structured SgrS is an important mechanism by which Hfq promotes the base pairing.


Assuntos
Pareamento de Bases , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Biossíntese de Proteínas , RNA Bacteriano/genética , Sequência de Bases , Regulação para Baixo , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/química , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , RNA Bacteriano/metabolismo
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