Sacerdot C, Chiaruttini C, Engst K, Graffe M, Milet M, Mathy N, Dondon J, Springer M
UPR9073, Institut de Biologie Physico Chimique, Paris, France.
Mol Microbiol. 1996 Jul;21(2):331-46. doi: 10.1046/j.1365-2958.1996.6361359.x.
The expression of the infC gene encoding translation initiation factor IF3 is negatively autoregulated at the level of translation, i.e. the expression of the gene is derepressed in a mutant infC background where the IF3 activity is lower than that of the wild type. The special initiation codon of infC, AUU, has previously been shown to be essential for derepression in vivo. In the present work, we provide evidence that the AUU initiation codon causes derepression by itself, because if the initiation codon of the thrS gene, encoding threonyl-tRNA synthetase, is changed from AUG to AUU, its expression is also derepressed in an infC mutant background. The same result was obtained with the rpsO gene encoding ribosomal protein S15. We also show that derepression of infC, thrS, and rpsO is obtained with other 'abnormal' initiation codons such as AUA, AUC, and CUG which initiate with the same low efficiency as AUU, and also with ACG which initiates with an even lower efficiency. Under conditions of IF3 excess, the expression of infC is repressed in the presence of the AUU or other 'abnormal' initiation codons. Under the same conditions and with the same set of 'abnormal' initiation codons, the repression of thrS and rpsO expression is weaker. This result suggests that the infC message has specific features that render its expression particularly sensitive to excess of IF3. We also studied another peculiarity of the infC message, namely the role of a GC-rich sequence located immediately downstream of the initiation codon and conserved through evolution. This sequence was proposed to interact with a conserved region in 16S RNA and enhance translation initiation. Unexpectedly, mutating this GC-rich sequence increases infC expression, indicating that this sequence has no enhancing role. Chemical and enzymatic probing of infC RNA synthesized in vitro indicates that this GC-rich sequence might pair with another region of the mRNA. On the basis of our in vivo results we propose, as suspected from earlier in vitro results, that IF3 regulates the expression of its own gene by using its ability to differentiate between 'normal' and 'abnormal' initiation codons.
编码翻译起始因子IF3的infC基因的表达在翻译水平上受到负向自动调节，即该基因的表达在IF3活性低于野生型的突变infC背景中去阻遏。infC的特殊起始密码子AUU此前已被证明对体内去阻遏至关重要。在本研究中，我们提供证据表明AUU起始密码子自身会导致去阻遏，因为如果编码苏氨酰 - tRNA合成酶的thrS基因的起始密码子从AUG变为AUU，其表达在infC突变背景中也会去阻遏。编码核糖体蛋白S15的rpsO基因也得到了相同的结果。我们还表明，infC、thrS和rpsO的去阻遏可通过其他“异常”起始密码子实现，如与AUU起始效率相同低的AUA、AUC和CUG，以及起始效率更低的ACG。在IF3过量的条件下，infC的表达在存在AUU或其他“异常”起始密码子的情况下受到抑制。在相同条件下且使用相同的“异常”起始密码子组，thrS和rpsO表达的抑制作用较弱。这一结果表明infC信使RNA具有使其表达对IF3过量特别敏感的特定特征。我们还研究了infC信使RNA的另一个特性，即位于起始密码子下游紧邻且在进化过程中保守的富含GC序列的作用。该序列被认为与16S RNA中的一个保守区域相互作用并增强翻译起始。出乎意料的是，突变这个富含GC的序列会增加infC的表达，表明该序列没有增强作用。对体外合成的infC RNA进行化学和酶促探测表明，这个富含GC的序列可能与mRNA的另一个区域配对。基于我们的体内结果，正如早期体外结果所推测的那样，我们提出IF3通过利用其区分“正常”和“异常”起始密码子的能力来调节其自身基因的表达。
