Light regulated translational activators: identification of chloroplast gene specific mRNA binding proteins.

Genetic analysis has revealed a set of nuclear-encoded factors that regulate chloroplast mRNA translation by interacting with the 5' leaders of chloroplastic mRNAs. We have identified and isolated proteins that bind specifically to the 5' leader of the chloroplastic psbA mRNA, encoding the photosystem II reaction center protein D1. Binding of these proteins protects a 36 base RNA fragment containing a stem-loop located upstream of the ribosome binding site. Binding of these proteins to the psbA mRNA correlates with the level of translation of psbA mRNA observed in light- and dark-grown wild type cells and in a mutant that lacks D1 synthesis in the dark. The accumulation of at least one of these psbA mRNA-binding proteins is dependent upon chloroplast development, while its mRNA-binding activity appears to be light modulated in developed chloroplasts. These nuclear encoded proteins are prime candidates for regulators of chloroplast protein synthesis and may play an important role in coordinating nuclear-chloroplast gene expression as well as provide a mechanism for regulating chloroplast gene expression during development in higher plants.     

小米psbA基因5''末端非编码区的结构特征

以小米(Setaria italica)为材料,克隆了含有叶绿体psbA基因的2.2kb EcoRⅠ片段,测定了该基因5＇末端非编码区的核苷酸序列。序列分析显示psbA基因5＇末端非编码区存在着与原核类似的启动子结构,其“-10”区的序列为TATACT,与原核生物“-10”共有基序(Consensus motif)仅相差一个核苷酸;其“-35”区的序列为TTGACA,与原核生物“-35”共有基序完全相同。另外,在“-10”区和“-35”区之间还存在着一个类似真核启动子结构的“TATATA”保守序列。这些结果表明小米psbA基因的启动子既具有原核的特征又具有真核的特征。小米psbA基因的mRNA前导序列长87bp,与高粱完全一致,而比水稻多出了“CTATTTT”7个核苷酸,比小麦、大麦和黑麦多出了“TTTT”4个核苷酸。因此推测在禾本科的C_3和C_4植物之间,psbA基因mRNA前导序列区的差异可能具有普遍性。计算机分析结果显示,以上6种植物的psbA基因mRNA前导序列区内均能形成小的茎环结构,而且这段“CTATTTT”额外序列恰好位于茎环结构中,造成了6种植物间茎环大小的差异。这一小的二级结构可能对psbA基因的表达调控有一定的影响。     

Accumulation of D1 polypeptide in tobacco plastids is regulated via the untranslated region of the psbA mRNA.

The plastid psbA mRNA is present in all tissues, while the encoded 32 kDa D1 protein of photosystem II accumulates tissue-specifically and in response to light. To study the regulation of D1 accumulation, a chimeric uidA gene encoding beta-glucuronidase (GUS) under control of the psbA 5'- and 3'-regulatory regions (224 and 393 bp, respectively), was integrated into the tobacco plastid genome. A high level of GUS accumulation in leaves and the lack of GUS in roots, with uidA mRNA present in both tissues, indicated tissue-specific accumulation of the chimeric gene product. Light-regulated accumulation of GUS in seedlings was shown. (i) Light-induced accumulation (100-fold) of GUS in etiolated cotyledons was accompanied by only a modest increase in mRNA levels. (ii) Inhibition of GUS synthesis was observed in cotyledons when light-grown seedlings were transferred to the dark, with no reduction in mRNA levels. Tissue-specific and light-regulated accumulation of GUS indicates that D1 accumulation is controlled via cis-acting regulatory elements in the untranslated region of the psbA mRNA. We propose that in tobacco, control of translation initiation is the primary mechanism regulating D1 protein accumulation.     

Translation of the psbA mRNA of Chlamydomonas reinhardtii requires a structured RNA element contained within the 5' untranslated region

Translational regulation is a key modulator of gene expression in chloroplasts of higher plants and algae. Genetic analysis has shown that translation of chloroplast mRNAs requires nuclear-encoded factors that interact with chloroplastic mRNAs in a message-specific manner. Using site-specific mutations of the chloroplastic psbA mRNA, we show that RNA elements contained within the 5' untranslated region of the mRNA are required for translation. One of these elements is a Shine- Dalgarno consensus sequence, which is necessary for ribosome association and psbA translation. A second element required for high levels of psbA translation is located adjacent to and upstream of the Shine-Dalgarno sequence, and maps to the location on the RNA previously identified as the site of message-specific protein binding. This second element appears to act as a translational attenuator that must be overcome to activate translation. Mutations that affect the secondary structure of these RNA elements greatly reduce the level of psbA translation, suggesting that secondary structure of these RNA elements plays a role in psbA translation. These data suggest a mechanism for translational activation of the chloroplast psbA mRNA in which an RNA element containing the ribosome-binding site is bound by message- specific RNA binding proteins allowing for increased ribosome association and translation initiation. These elements may be involved in the light-regulated translation of the psbA mRNA.     

Structural feature of sorghum chloroplast psbA gene and regulation effects of its 5'-noncoding region*

Comparative analysis reveals remarkable homology between the sequences of both psbA gene nucleotides and the inferred amino acids of sorghum, a C_4 plant, and those of rice, a C_3 plant. The 5'-noncoding region of sorghum psbA gene contains the conservative promoter elements, "-35" element and "-10" element, like the prokaryote and the promoter element, TATA box, like the eukaryote. As compared with that of the rice, an extra sequence of 7 bp is found in the leader sequence of the mRNA in the former. Using an in vitro system, it has been demonstrated that protein factor exists in sorghum chloroplast protein extract which specifically binds to the 5'-noncoding region of psbA gene. Measurement of the expression of luciferase shows a 2—5 time greater reaction of the expression plasmids pALqs which contain leader region of sorghum psbA gene than that of the expression plasmids pALqr which contain leader region of rice psbA gene in E. coli.     

The 5' untranslated region of the maize alcohol dehydrogenase gene provides efficient translation of mRNA in plants under stress conditions

Reduced level of expression of most cell proteins under stress conditions is determined by low efficiency of cap-dependent translation of corresponding mRNAs. The maize gene encoding alcohol dehydrogenase, adh1, is an example of a gene which mRNA is efficiently translated under hypoxia. Using reporter gene assay we showed that the leader sequence of adh1 mRNA, provides efficient translation of reporter gene gfp in Nicotiana benthamiana cells under hypoxia and heat shock. The presence of this leader sequence in 5' UTR of mRNA does not change the level of expression in aerobic conditions, but under hypoxia and heat shock the levels of reporter gfp expression were reduced about 5-10 fold in the absence of leader and remained unaffected in its presence in 5'UTR. We found that this leader sequence does not change the level of mRNA stability and does not exhibit promoter activity. Consequently, leader sequence acts as translational enhancer providing efficient translation of mRNA in plant cells under stress conditions. Introduction of this sequence into standard expression cassettes may be used for development of new systems of expression of target proteins in plants, efficient under stress conditions.     

Structural Analysis of rbcL Gene from an Endangered Plant，（Acanthopanax brachypus）

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Regulation of rat ornithine decarboxylase mRNA translation by its 5'-untranslated region

Ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, is a highly inducible protein whose expression involves a complex and variable array of regulatory mechanisms. We investigated the influence of the 5'-untranslated region (5'UTR) of the rat ODC mRNA on translation of the mRNA in a cell-free system and in cultured mammalian cells. ODC mRNA containing the full-length 5'UTR was translated in reticulocyte lysates at approximately 5% of the rate of mRNA containing no ODC 5' leader sequences. The complete 5'UTR inhibited expression of a heterologous gene product, human growth hormone, to the same extent in cultured mammalian cells. Furthermore, the 5'-most 130 bases of the rat ODC 5'UTR, a conserved G/C-rich region predicted to form a stable stem-loop structure (delta G = -68 kcal/mol), repressed translation to the same extent as the entire 5'UTR, both in the lysates and in intact cells. The 3'-most 160 bases of the 5'UTR, containing a small upstream open reading frame, decreased expression by 50-65% both in vitro and in intact cells, compared with controls lacking any ODC 5'UTR sequences. Mutation of the initiation codon AUG beginning this upstream open reading frame to GCG restored expression to rates equivalent to those seen in constructions containing no ODC 5'UTR sequences. We conclude that the rat ODC mRNA 5'UTR can inhibit translation of ODC mRNA both in vitro and in vivo, and that the predicted stem-loop structure at the 5' end of the 5'UTR is both necessary and sufficient for this inhibition.     

水稻叶绿体16S启动子克隆改造、载体构建及转化研究

利用PCR方法从水稻叶绿体基因组DNA中分离16S启动子,并在其下游加入rbcL基因SD序列,以增强该启动子的翻译能力;序列分析表明,除加入的SD序列外,扩增片段与水稻(Oryza sativa)叶绿体基因组DNA序列16S启动子相应区域同源性为100％。将16S启动子与bar基因和gfp基因的融合基因连接,以psbA基因的3′序列为终止子,并以烟草叶绿体trnH—psbA和trnK为同源片段构建了烟草叶绿体表达载体pRl6S。用基因枪转化烟草,转化植株经Southern、Northern检测及后代遗传学分析,发现：16S启动子具有启动活性,融合基因已在烟草叶绿体中稳定整合并遵循母系遗传规律。     

Importance of the leader region of mRNA for translation initiation of ColE2 Rep protein

Translation initiation of mRNA encoding the Rep protein of the ColE2 plasmid required for initiation of plasmid DNA replication is fairly efficient in Escherichia coli cells despite the absence of a canonical Shine-Dalgarno sequence. To define sequences and structural elements responsible for translation efficiency of the Rep mRNA, a series of rep-lacZalpha translational fusions bearing various mutations in the region encoding the leader region of the Rep mRNA was generated and tested for the translation activity by measuring the beta-galactosidase activity. We showed that the region rich in A and U between the stem-loop II structure and GA cluster sequence, formation of the stem-loop II structure, but not its sequence, and the region between the GA cluster sequence and initiation codon are important along with the GA cluster sequence for efficient translation of the Rep protein. The existence of these important regions in the leader region of the Rep mRNA may explain the mechanism of inhibition of the Rep protein translation by an antisense RNA (RNAI), which is complementary to the leader region.