The cysteine desulfurase IscS is required for synthesis of all five thiolated Nucleosides present in tRNA from Salmonella enterica serovar Typhimurium
The cysteine desulfurase IscS is required for synthesis of all five thiolated Nucleosides present in tRNA from Salmonella enterica serovar Typhimurium
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DOI：
10.1128/jb.184.24.6830-6835.2002
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发表时间：
2002-12-01
影响因子：
3.2
通讯作者：
Björk, GR
中科院分区：
文献类型：
--
作者：
Nilsson, K;Lundgren, HK;Björk, GR
tRNA中修饰核苷的缺乏通常介导+1移码突变的抑制。在需要组氨酸才能生长的肠沙门氏菌血清型鼠伤寒菌TR970 （hisC3737）中，一个潜在的+1移框位点CCC-CAA-UAA存在于hisC基因中插入C所产生的移框窗口内。该位点可能被肽基trnap (cmo5UGG)(Pro) （cmo(5)U为尿嘧啶-5-氧乙酸）抑制，在解码近同源密码子CCC时产生移码，如果tRNA(mnm5s2UUG)(Gln) (mnm（5)s(2)U为5-甲基胺甲基-2-硫脲）缓慢进入位于a位点的CAA密码子时发生停顿。我们选择了TR970菌株能够在没有组氨酸的情况下生长的突变体，其中一个突变体（iscS51）被证明在l -半胱氨酸脱硫酶IscS中有氨基酸取代。此外，在iscS51突变体中，肠链球菌tRNA中存在的所有5种硫代核苷2-硫代胞苷、mnm(5)s(2)U、5-羧甲基氨基甲基-2-硫代嘧啶、4-硫代嘧啶和N-6-（4-羟基异戊烯基）-2-甲基硫代腺苷的水平都降低了。在对数生长的大肠杆菌细胞中，iscS基因的缺失导致tRNA中除N-6-（4-羟基异戊烯基）-2-甲基硫代腺苷外的所有硫代核苷含量均检测不到，其含量仅为野生型的1.6%。细胞在固定期长时间孵育后，观察到20%水平的2-硫代胞苷和2%水平的N-6-（4-羟基异戊烯基）-2-甲基硫代腺苷，而没有发现4-硫代嘧啶、5-羧甲基胺甲基-2-硫代嘧啶或mnm(5)s(2)U。我们将iscS51突变介导的移帧能力归因于tRNA(mnm5s2UGG)（Gln）由于mnm(5)s(2) U缺乏而导致CAA解码缓慢。由于iscS缺失突变体在富培养基中的生长速度与只缺乏mnm(5)s(2)U的突变体（mmnA）相似，我们认为iscS缺失突变体生长速度降低的主要原因是缺乏mnm(5)s(2)U和5-羧甲基胺甲基-2-硫脲，而不是缺乏其他三种硫代核苷，这些核苷在iscS缺失突变体中也不存在。
Deficiency of a modified nucleoside in tRNA often mediates suppression of +1 frameshift mutations. In Salmonella enterica serovar Typhimurium strain TR970 (hisC3737), which requires histidine for growth, a potential +1 frameshifting site, CCC-CAA-UAA, exists within the frameshifting window created by insertion of a C in the hisC gene. This site may be suppressed by peptidyl-tRNAP(cmo5UGG)(Pro) (cmo(5)U is uridine-5-oxyacetic acid), making a frameshift when decoding the near-cognate codon CCC, provided that a pause occurs by, e.g., a slow entry of the tRNA(mnm5s2UUG)(Gln) (mnm(5)s(2)U is 5-methylaminomethyl-2-thiouridine) to the CAA codon located in the A site. We selected mutants of strain TR970 that were able to grow without histidine, and one such mutant (iscS51) was shown to have an amino acid substitution in the L-cysteine desulfurase IscS. Moreover, the levels of all five thiolated nucleosides 2-thiocytidine, mnm(5)s(2)U, 5-carboxymethylaminomethyl-2-thiouridine, 4-thiouridine, and N-6-(4-hydroxyisopentenyl)-2-methylthioadenosine present in the tRNA of S. enterica were reduced in the iscS51 mutant. In logarithmically growing cells of Escherichia coli, a deletion of the iscS gene resulted in nondetectable levels of all thiolated nucleosides in tRNA except N-6-(4-hydroxyisopentenyl)-2-methylthioadenosine, which was present at only 1.6% of the wild-type level. After prolonged incubation of cells in stationary phase, a 20% level of 2-thiocytidine and a 2% level of N-6-(4-hydroxyisopentenyl)-2-methylthioadenosine was observed, whereas no 4-thiouridine, 5-carboxymethylaminomethyl-2-thiouridine, or mnm(5)s(2)U was found. We attribute the frameshifting ability mediated by the iscS51 mutation to a slow decoding of CAA by the tRNA(mnm5s2UGG)(Gln) due to mnm(5)s(2) U deficiency. Since the growth rate of the iscS deletion mutant in rich medium was similar to that of a mutant (mmnA) lacking only mnm(5)s(2)U, we suggest that the major cause for the reduced growth rate of the iscS deletion mutant is the lack of mnm(5)s(2) U and 5-carboxymethylaminomethyl-2-thiouridine and not the lack of any of the other three thiolated nucleosides that are also absent in the iscS deletion mutant.