Resolution of Two Factors required in the Qβ-RNA Polymerase Reaction

Shapiro, Lucille; Fernández, M. T. Franze de; August, J. T.

doi:10.1038/220478a0

Cited by 60 publications

(14 citation statements)

References 4 publications

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“…Hfq was first discovered in the mid-1960s in a screen for host proteins required for replication of Q-beta bacteriophage in Escherichia coli , 1972Shapiro et al 1968). The physiological role of this highly conserved RNA binding protein was unclear at the time.…”

Section: Introductionmentioning

confidence: 99%

The RNA binding protein Hfq interacts specifically with tRNAs

Lee

Feig²

2008

RNA

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Hfq is an RNA binding protein that has been studied extensively for its role in the biology of small noncoding RNAs (ncRNAs) in bacteria, where it facilitates post-transcriptional gene regulation during stress responses. We show that Hfq also binds with high specificity and nanomolar affinity to tRNAs despite their lack of a canonical A/U rich single-stranded sequence. This affinity is comparable to that of Hfq for its validated ncRNA targets. Two sites on tRNAs are protected by Hfq binding, one on the D-stem and the other on the T-stem. Mutational analysis and competitive binding experiments indicate that Hfq uses its proximal surface (also called the L4 face) to bind tRNAs, the same surface that interacts with ncRNAs but a site distinct from where poly(A) oligonucleotides bind. hfq knockout strains are known to have broad pleiotropic phenotypes, but none of them are easily explained by or imply a role for tRNA binding. We show that hfq deletion strains have a previously unrecognized phenotype associated with mistranslation and significantly reduced translational fidelity. We infer that tRNA binding and reduced fidelity are linked by a role for Hfq in tRNA modification.

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Section: Introductionmentioning

confidence: 99%

The RNA binding protein Hfq interacts specifically with tRNAs

Lee

Feig²

2008

RNA

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“…The basic replication machinery is composed of one phage-encoded protein (subunit 1B) and three host-encoded proteins, i.e., 30S ribosome protein S1 (subunit et) and protein synthesis elongation factors EF-Tu (subunit -y) and EF-Ts (subunit 6) (1). In addition, the synthesis of cRNA (the first-step reaction of RNA replication) requires an Escherichia coli protein which has hitherto been referred to as host factor I (HF-I) (4,6,19). In the absence of this factor, the initiation of cRNA synthesis does not take place at all (7,14), while the cRNA-directed vRNA synthesis proceeds in its absence.…”

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confidence: 99%

Regulation of the Escherichia coli hfq gene encoding the host factor for phage Q beta

et al. 1994

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The host factor (HF-I) for phage Q beta RNA replication is a small protein of 102 amino acid residues encoded by the hfq gene at 94.8 min on the Escherichia coli chromosome. The synthesis rate of HF-I at the exponential-growth phase is higher than at the stationary phase, and it increases concomitantly with the increase in cell growth rate. The intracellular level of HF-I is about 30,000 to 60,000 molecules per cell, the majority being associated with ribosomes as one of the salt wash proteins. Taken together, we suggest that HF-I is one of the growth-related proteins.

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“…A strong candidate cofactor was Hfq, an Escherichia coli protein required for replication of the RNA genome of the Q␤ phage both in vivo and in vitro (7,8,23). Hfq functions by destabilizing an RNA secondary structure on the 3Ј end of the positive strand of Q␤ (7,8,23). In addition, Hfq binds tightly to Q␤ RNA, poly(A) RNA (4,8,22), and OxyS RNA (37).…”

mentioning

confidence: 99%

“…Since many other RNAs require protein cofactors for activity, we looked for proteins that might be necessary for the DsrA-mediated regulation. A strong candidate cofactor was Hfq, an Escherichia coli protein required for replication of the RNA genome of the Q␤ phage both in vivo and in vitro (7,8,23). Hfq functions by destabilizing an RNA secondary structure on the 3Ј end of the positive strand of Q␤ (7,8,23).…”

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confidence: 99%

Hfq Is Necessary for Regulation by the Untranslated RNA DsrA

2001

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DsrA is an 85-nucleotide, untranslated RNA that has multiple regulatory activities at 30°C. These activities include the translational regulation of RpoS and H-NS, global transcriptional regulators in Escherichia coli. Hfq is an E. coli protein necessary for the in vitro and in vivo replication of the RNA phage Q␤. Hfq also plays a role in the degradation of numerous RNA transcripts. Here we show that an hfq mutant strain is defective for DsrA-mediated regulation of both rpoS and hns. The defect in rpoS expression can be partially overcome by overexpression of DsrA. Hfq does not regulate the transcription of DsrA, and DsrA does not alter the accumulation of Hfq. However, in an hfq mutant, chromosome-expressed DsrA was unstable (half-life of 1 min) and truncated at the 3 end. When expressed from a multicopy plasmid, DsrA was stable in both wild-type and hfq mutant strains, but it had only partial activity in the hfq mutant strain. Purified Hfq binds DsrA in vitro. These results suggest that Hfq acts as a protein cofactor for the regulatory activities of DsrA by either altering the structure of DsrA or forming an active RNA-protein complex.

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Resolution of Two Factors required in the Qβ-RNA Polymerase Reaction

Cited by 60 publications

References 4 publications

The RNA binding protein Hfq interacts specifically with tRNAs

The RNA binding protein Hfq interacts specifically with tRNAs

Regulation of the Escherichia coli hfq gene encoding the host factor for phage Q beta

Hfq Is Necessary for Regulation by the Untranslated RNA DsrA

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