Suppression of the Escherichia coli rpoH opal mutation by ribosomes lacking S15 protein.

Several suppressors (suhD) that can specifically suppress the temperature-sensitive opal rpoH11 mutation of Escherichia coli K-12 have been isolated and characterized. Unlike the parental rpoH11 mutant deficient in the heat shock response, the temperature-resistant pseudorevertants carrying suhD were capable of synthesizing sigma 32 and exhibiting partial induction of heat shock proteins. These strains were also cold sensitive and unable to grow at 25 degrees C. Genetic mapping and complementation studies permitted us to localize suhD near rpsO (69 min), the structural gene for ribosomal protein S15. Ribosomes and polyribosomes prepared from suhD cells contained a reduced level (ca. 10%) of S15 relative to that of the wild type. Cloning and sequencing of suhD revealed that an IS10-like element had been inserted at the attenuator-terminator region immediately downstream of the rpsO coding region. The rpsO mRNA level in the suhD strain was also reduced to about 10% that of wild type. Apparently, ribosomes lacking S15 can actively participate in protein synthesis and suppress the rpoH11 opal (UGA) mutation at high temperature but cannot sustain cell growth at low temperature.     

Specific recognition of rpsO mRNA and 16S rRNA by Escherichia coli ribosomal protein S15 relies on both mimicry and site differentiation

The ribosomal protein S15 binds to 16S rRNA, during ribosome assembly, and to its own mRNA (rpsO mRNA), affecting autocontrol of its expression. In both cases, the RNA binding site is bipartite with a common subsite consisting of a G*U/G-C motif. The second subsite is located in a three-way junction in 16S rRNA and in the distal part of a stem forming a pseudoknot in Escherichia coli rpsO mRNA. To determine the extent of mimicry between these two RNA targets, we determined which amino acids interact with rpsO mRNA. A plasmid carrying rpsO (the S15 gene) was mutagenized and introduced into a strain lacking S15 and harbouring an rpsO-lacZ translational fusion. Analysis of deregulated mutants shows that each subsite of rpsO mRNA is recognized by a set of amino acids known to interact with 16S rRNA. In addition to the G*U/G-C motif, which is recognized by the same amino acids in both targets, the other subsite interacts with amino acids also involved in contacts with helix H22 of 16S rRNA, in the region adjacent to the three-way junction. However, specific S15-rpsO mRNA interactions can also be found, probably with A(-46) in loop L1 of the pseudoknot, demonstrating that mimicry between the two targets is limited.     

Expression of the rpsO and pnp genes: structural analysis of a DNA fragment carrying their control regions.

Precise physical mapping of the genes rpsO and pnp coding respectively for ribosomal protein S15 and polynucleotide phosphorylase together with regions involved in the regulation of their expression has been obtained by the analysis of in vitro deletion mutants. The results suggest that each gene has its own promotor, but that there is coexpression of rpsO and pnp. The nucleotide sequence of rpsO and of the beginning of pnp is presented and includes the presumed regulatory regions of these genes. Several features of the sequence support the mapping experiments and are discussed in relation to the expression of the ribosomal and pnp genes.     

Human polynucleotide phosphorylase,hPNPase, is localized in mitochondria

The human gene encoding a polynucleotide phosphorylase (hPNPase) has been recently identified as strongly up-regulated in two processes leading to irreversible arrest of cell division: progeroid senescence and terminal differentiation. Here, we demonstrate that the hPNPase is localized in mitochondria. Our finding suggests the involvement of mitochondrial RNA metabolism in cellular senescence.     

大肠杆菌嘌呤核苷磷酸化酶基因的克隆与真核表达载体的构建

根据Ｇｅｎｂａｎｋ中大肠杆菌嘌呤核苷磷酸化酶（ＰＮＰ）基因的核苷酸序列,设计并合成了一对引物,以大肠杆菌基因组ＤＮＡ为模板,进行ＰＣＲ扩增,并将扩增产物定向连接到克隆、测序及真核表达载体ＰＣＤＮＡ３中,进行酶切鉴定、测序及序列分析。结果表明ＰＣＲ扩增出７４１ｂｐ大小的片段,通过酶切和序列分析证明含完整的ＰＮＰ基因序列且基因插入方向正确,此序列与文献报道的ＰＮＰ基因的同源性为９９．７％。说明克隆的ＰＮＰ基因与文献报道的基本一致,ｐｃＤＮＡ３－ＰＮＰ的构建成功为今后用其进行基因转染来研究ＰＮＰ／Ｍｅｐ－ｄＲ自杀基因系统在肿瘤基因治疗中的应用打下了基础。