Updegrove Taylor B, Wartell Roger M
School of Biology, Georgia Institute of Technology, Atlanta, GA 30332, USA.
Biochim Biophys Acta. 2011 Oct;1809(10):532-40. doi: 10.1016/j.bbagrm.2011.08.006. Epub 2011 Aug 22.
The Escherichia coli RNA binding protein Hfq plays an important role in regulating mRNA translation through its interactions with small non-coding RNAs (sRNAs) and specific mRNAs sites. The rpoS mRNA, which codes for a transcription factor, is regulated by several sRNAs. DsrA and RprA enhance translation by pairing to a site on this mRNA, while OxyS represses rpoS mRNA translation. To better understand how Hfq interacts with these sRNAs and rpoS mRNA, the binding of wt Hfq and eleven mutant Hfqs to DsrA, RprA, OxyS and rpoS mRNA was examined. Nine of the mutant Hfq had single-residue mutations located on the proximal, distal, and outer-edge surfaces of the Hfq hexamer, while two Hfq had truncated C-terminal ends. Hfq with outer-edge mutations and truncated C-terminal ends behaved similar to wt Hfq with regard to binding the sRNAs, rpoS mRNA segments, and stimulating DsrA•rpoS mRNA formation. Proximal surface mutations decreased Hfq binding to the three sRNAs and the rpoS mRNA segment containing the translation initiation region. Distal surface mutations lowered Hfq's affinity for the rpoS mRNA segment containing the (ARN)(4) sequence. Strong Hfq binding to both rpoS mRNA segments appears to be needed for maximum enhancement of DsrA•rpoS mRNA annealing. OxyS bound tightly to Hfq but exhibited weak affinity for rpoS mRNA containing the leader region and 75 nt of coding sequence in the absence or presence of Hfq. This together with other results suggest OxyS represses rpoS mRNA translation by sequestering Hfq rather than binding to rpoS mRNA.
大肠杆菌RNA结合蛋白Hfq通过与小非编码RNA（sRNA）和特定mRNA位点相互作用，在调节mRNA翻译中发挥重要作用。编码转录因子的rpoS mRNA受多种sRNA调控。DsrA和RprA通过与该mRNA上的一个位点配对来增强翻译，而OxyS则抑制rpoS mRNA的翻译。为了更好地理解Hfq如何与这些sRNA和rpoS mRNA相互作用，研究了野生型Hfq和11种突变型Hfq与DsrA、RprA、OxyS和rpoS mRNA的结合情况。其中9种突变型Hfq在Hfq六聚体的近端、远端和外缘表面有单残基突变，另外两种Hfq的C末端被截断。具有外缘突变和C末端截断的Hfq在结合sRNA、rpoS mRNA片段以及刺激DsrA•rpoS mRNA形成方面的表现与野生型Hfq相似。近端表面突变降低了Hfq与三种sRNA以及包含翻译起始区域的rpoS mRNA片段的结合。远端表面突变降低了Hfq对包含（ARN）(4)序列的rpoS mRNA片段的亲和力。Hfq与两个rpoS mRNA片段的强结合似乎是最大程度增强DsrA•rpoS mRNA退火所必需的。在不存在或存在Hfq的情况下，OxyS与Hfq紧密结合，但对包含前导区和75个核苷酸编码序列的rpoS mRNA表现出弱亲和力。这与其他结果一起表明，OxyS通过隔离Hfq而非与rpoS mRNA结合来抑制rpoS mRNA的翻译。
