mRNA翻译起始区构效关系的研究

本文分析了9个表达量相差55倍的5′PCNA-LacZ′重组子的TIR二级结构与翻译起始效率的关系。在5′NTR顺序确定为28个核苷酸(从转录起始位点起)时,TIR范围在约编码第12个密码子(第33至37核苷酸)处存在一个二级结构及其△G的变化阈位,在该阈位处按表达量调正重组子TIR的范围,得到△G与表达量之间相关系数为0.97的曲线;在TIR二级结构中,起始密码子处于配对的空间位置对翻译起始效率有明显抑制作用,但SD顺序的空间位置与表达量之间无明显相关性。     

降低mRNA翻译起始区的稳定性原核非融合表达HAb18GEF

为在大肠杆菌中非融合表达肝癌相关抗原HAb18G胞外区片段（HAb18GEF）,将HAb18GEF基因的cDNA插入原核表达载体pET21a+。通过计算机辅助设计,对重组的HAb18GEF/pET21a+的mRNA翻译起始区（TIR）的二级结构和密码子偏性同时进行预测。结果发现其存在稳定的茎环结构和许多稀有密码子。通过优化二级结构和优化密码子偏性二种策略分别来降低HAb18GEF/pET21a+的mRNA翻译起始区（TIR）的稳定性。在不改变氨基酸序列的前提下,利用密码子的简并性,通过非连续定点突变实现这两种优化。将突变前后的重组子经酶切鉴定和测序验证后,转化感受态JM109DE3宿主菌后,随机挑菌37℃下用IPTG诱导表达。SDSPAGE、间接ELISA、Western blot 和细胞分级分离法分析这些重组子的诱导表达情况。RNA dot blot对比分析优化前后目的基因mRNA的量。结果证明,成功地构建了HAb18GEF/pET21a+及其二种优化突变体。仅优化TIR区二级结构或仅优化TIR区密码子偏性均能实现HAb18GEF蛋白的非融合表达,而未优化的重组子不表达任何HAb18GEF。非融合表达产物在大肠杆菌中主要以包涵体形式存在,高达293%。由于过表达和细胞渗漏,培养基和周质腔中也可检测到少许的HAb18GEF。优化二级结构和优化密码子偏性二种策略的HAb18GEF的非融合表达量基本相同。优化前后HAb18GEF转录的mRNA量没有差别。这些结果表明,降低mRNA翻译起始区的稳定性可实现肝癌相关抗原HAb18G胞外区片段在大肠杆菌中的非融合表达。     

人α－肿瘤坏死因子基因的高效表达

用人工合成的α－肿瘤坏死因子(TNF—α)基因构建了五个不同的表达质粒,它们不同之处是SD序列与起始密码子ATG问距离(D)各异．计算机模拟计算出翻译起始区域(TIR)中二级结构的最小生成自由能．以(D)为6个核苷酸时最小(绝对值)。它的表达效率也最高,产物TNF—α可达菌体总蛋白的60％．密码子的选用对表达效率有很大的影响,故人工合成TNF－α基因(选用大肠杆菌偏爱的密码子)的表达效率高于sc－DNA(对部分密码子改造的半合成eDNA)．     

Mx基因稀有密码子和mRNA结构及大肠杆菌表达 优化

通过对稀有密码子和mRNA翻译起始区二级结构的分析, 构建了4种重组表达菌株BL21(DE3)/pET-Mx, Rosseta(DE3)/pET-Mx, BL21(DE3)/pGEX-Mx和Rosseta(DE3)/pGEX-Mx, 在大肠杆菌中进行Mx基因的表达, Rosseta(DE3)/pET-Mx和Rosseta(DE3)/pGEX-Mx重组表达菌中都获得了表达, Western blotting检测到了特异的75 kDa表达产物。实验结果证明稀有密码子和mRNA翻译起始区二级结构对Mx 蛋白表达都有影响, 选择适用于稀有密码子表达的菌株Rosetta(DE3)有利于Mx蛋白的表达, 同时翻译起始区二级结构能值较低的表达载体pGEX-Mx获得的表达量明显增高。实验中首次获得了重组表达鸡全长Mx蛋白的大肠杆菌重组菌。     

鸡基因组差异表达基因翻译起始密码子上下游序列的比较研究

翻译起始调控是基因表达调控的一个关键步骤之一。本文以鸡为研究材料,比较研究了鸡基因组高表达基因和低表达基因翻译起始密码子上下游的碱基序列差异,旨在寻找影响鸡基因表达水平的特异性调控位点。全部3 020个单剪接基因完整的mRNA序列及有详细注释的5＇UTRs序列从Ensembl下载。编写计算机程序,读取每个基因mRNA起始密码子上下游各位点的碱基。研究发现,起始密码子上游-3、-2位点可能是鸡基因组基因表达起始密码子正确识别的关键位点。起始密码子上下游的碱基组成分析发现,高表达基因和低表达基因起始密码子的上游均倾向使用（G＋C）,高表达基因的使用偏倚尤为强烈。序列差异比较发现,高表达基因在-9、-6、-3、＋4位点显著偏向G,在-1、-2、-4、-5位点显著偏向C。低表达基因起始密码子上游使用A、U的频率显著高于低表达基因。在-19位点强烈偏向A,在＋1、＋11、＋14位点强烈偏向U。     

基于mRNA 5'端TIR区二级结构优化提高重组sTNFαRI在大肠杆菌中的表达水平

目的:通过优化PET11b-s TNFαRI 5'mRNA翻译起始区(TIR)二级结构从而提高可溶性肿瘤坏死因子I型受体(sTNFαRI)在大肠杆菌[E.coli BL21(DE3)]中的表达水平。方法:通过对PET11b-s TNFαRI mRNA 5'端TIR区二级结构的自由能及核苷酸位置熵分析,设计相应的引物对mRNA 5'翻译起始区(TIR)相应密码子进行突变,从而使核糖体结合位点(RBS)及起始密码子(AUG)暴露于发夹结构之外,此外将p ET11b核糖体结合位点由GAAGGAGA突变为GAAGAA,以利于翻译复合体的组装以及翻译起始。通过基因克隆的方法将5'端TIR区优化后的序列与s TNFαRI序列一起克隆到p ET11b载体中,并转化大肠杆菌BL21(DE3),阳性转化子经IPTG诱导表达,SDS-PAGE和Western blot检测。结果:通过对PET11b-s TNFαRI 5'TIR mRNA二级结构优化,经SDS-PAGE和Western blot分析表明重组s TNFαRI的表达水平较优化前提高50%~60%。结论:通过对重组载体翻译起始区(TIR)mRNA序列的二级结构优化可以有效提高目的蛋白的表达水平,对进一步工业化生产具有重要的应用价值。     

m RNA5′端不同位置的二级结构对原核生物翻译起始的影响

 一个 2 1 bp的序列插入 B50中的人 PCNA- Lac Z′融合蛋白 m RNA的 SD序列的上游 1 1 bpH ind 切点处 ,构成正、反向插入的两个重组质粒 B50 il、B50 i2 .通过计算机程序对 m RNA二级结构的模拟分析 ,B50 il的插入序列在翻译起始区 (TIR)前形成一个发夹结构 ,但不影响 TIR的二级结构 ;B50 i2的插入序列在 TIR 5′端形成一个二级结构 ;而另一克隆 D1 3与 B50因 SD前后的 6个和 7个碱基序列的不同 ;使翻译起始区 TIR的 SD到 AUG附近的二级结构不相同 .实验测定的四者的β-半乳糖苷酶活性与计算机计算的解开 m RNA TIR的二级结构所需能量ΔE进行比较 ,其结果说明 m RNA TIR前面的二级结构对翻译起始无影响 ,而位于 TIR的 5′端的二级结构对翻译起始效率是有影响的 .不过 ,它与 m RNA TIR的 SD到 AUG的附近的二级结构对翻译起始效率的影响相比 ,TIR5′端二级结构的影响比较小 .同时 ,PCNA- Lac Z和 PCNA- Lac Z′两种融合蛋白的β-半乳糖苷酶活性也进行了比较 ,酶活性有差别 ,但不同质粒酶活性的比值仍相同 .     

m RNA5′端不同位置的二级结构对原核生物翻译起始的影响

一个 2 1 bp的序列插入 B50中的人 PCNA- Lac Z′融合蛋白 m RNA的 SD序列的上游 1 1 bpH ind 切点处 ,构成正、反向插入的两个重组质粒 B50 il、B50 i2 .通过计算机程序对 m RNA二级结构的模拟分析 ,B50 il的插入序列在翻译起始区 (TIR)前形成一个发夹结构 ,但不影响 TIR的二级结构 ;B50 i2的插入序列在 TIR 5′端形成一个二级结构 ;而另一克隆 D1 3与 B50因 SD前后的 6个和 7个碱基序列的不同 ;使翻译起始区 TIR的 SD到 AUG附近的二级结构不相同 .实验测定的四者的β-半乳糖苷酶活性与计算机计算的解开 m RNA TIR的二级结构所需能量ΔE进行比较 ,其结果说明 m RNA TIR前面的二级结构对翻译起始无影响 ,而位于 TIR的 5′端的二级结构对翻译起始效率是有影响的 .不过 ,它与 m RNA TIR的 SD到 AUG的附近的二级结构对翻译起始效率的影响相比 ,TIR5′端二级结构的影响比较小 .同时 ,PCNA- Lac Z和 PCNA- Lac Z′两种融合蛋白的β-半乳糖苷酶活性也进行了比较 ,酶活性有差别 ,但不同质粒酶活性的比值仍相同 .     

红霉素合成蛋白基因的克隆和高表达

文献报道红霉素合成蛋白DEBS2 (6-脱氧红霉内酯B合酶)(374 kD)基因在大肠杆菌(Escherichia coli)中异源表达量比较低,难以开展后续蛋白纯化和结构等研究.为获得该大蛋白基因的异源高表达,本研究通过PCR的方法,对编码红霉素合成蛋白DEBS2的基因起始5'端设计了起始密码子之后的十一个简并密码子序列,随机筛选获得在表达宿主BL21(DE3)中高表达的简并序列质粒,质粒命名为DEBS2-17.结果 表明:mRNA的起始二级结构影响200 kD以上的蛋白质表达水平.本研究为开展红霉素合成蛋白的结构及生化研究提供理论基础,同时对表达分子量200 kD以上蛋白具有一定借鉴意义.     

使用一种新策略在大肠杆菌中高效表达hbFGF

翻译起始区（TIR）二级结构是影响翻译效率的决定性因素,同时密码子的偏好性问题也是个至关重要的方面。基于以上两点考虑,对hbFGF5‘末端35个碱基进行了改造,对其中4个位点进行了定点突变,另有4个位点进行了随机点突变。这些突变都可能造成TIR二级结构变化。这4个随机突变共有32种组合,使用RNA结构预测软件DNASIS v2.5对这32种序列分别模拟其二级结构且计算其自由能,并选取了10条自由能最高的序列。根据这10条序列,分别设计引物引入突变,克隆至表达载体pET-3c上,然后转化宿主菌E.coli,通过诱导表达纯化及生物测活等常规实验方法,最后确定有两株为高表达菌株,从某种程度上证明了用计算机辅助设计定点突变的方法来优化外源基因在E.coli中的表达是有效且很有潜力的。     

The dependence of the E.coli gene expression level on the structure of the translation initiation region (TIR). IV. Distal complementary TIR interactions with the mRNA coding region

To evaluate the effect on translation of distal regions of the encoding mRNA part capable of the complementary binding to the ribosome binding site (RBS), a series of plasmids were constructed containing fragments inserted into the il3 gene and determining secondary interactions in mRNA. A comparison of the levels of the in vivo gene expression showed that the complementary interactions of the translation initiation region (TIR) with distal regions of the mRNA encoding part affect translation. The effectiveness of these interactions decreased with an increase in the distance between the RBS and the complementary mRNA region, whereas the secondary structure formed by the TIR and the adjacent mRNA region was more stable despite the presence of regions in mRNA capable of forming energetically more favorable structures involving these elements.     

Kinetic checkpoint at a late step in translation initiation

The translation initiation efficiency of a given mRNA is determined by its translation initiation region (TIR). mRNAs are selected into 30S initiation complexes according to the strengths of the secondary structure of the TIR, the pairing of the Shine-Dalgarno sequence with 16S rRNA, and the interaction between initiator tRNA and the start codon. Here, we show that the conversion of the 30S initiation complex into the translating 70S ribosome constitutes another important mRNA control checkpoint. Kinetic analysis reveals that 50S subunit joining and dissociation of IF3 are strongly influenced by the nature of the codon used for initiation and the structural elements of the TIR. Coupling between the TIR and the rate of 70S initiation complex formation involves IF3- and IF1-induced rearrangements of the 30S subunit, providing a mechanism by which the ribosome senses the TIR and determines the efficiency of translational initiation of a particular mRNA.     

Translational stimulation: RNA sequence and structure requirements for binding of Com protein.

Translation of the bacteriophage Mu mom gene is positively regulated by the phage Com protein. We report here that purified Com protein specifically stimulates mom gene expression in vitro. Furthermore, Com is shown to bind a site in the mom translational initiation region (TIR) in a sequence-specific manner. In vitro RNA footprint experiments have been used to define the Com-binding site and to study mRNA secondary structure in the mom TIR. Com binding is shown to correlate with a conformational change in the mom TIR both in vivo and in vitro. The role of secondary structure was further examined by testing the effects of mutations in the TIR on translation and stimulation. The results support a model for translational stimulation in which Com binding induces a conformational change in the mom mRNA, thereby enhancing ribosome binding.     

Effects of different target sites on antisense RNA-mediated regulation of gene expression

Antisense RNA is a type of noncoding RNA (ncRNA) that binds to complementary mRNA sequences and induces gene repression by inhibiting translation or degrading mRNA. Recently, several small ncRNAs (sRNAs) have been identified in Escherichia coli that act as antisense RNA mainly via base pairing with mRNA. The base pairing predominantly leads to gene repression, and in some cases, gene activation. In the current study, we examined how the location of target sites affects sRNA-mediated gene regulation. An efficient antisense RNA expression system was developed, and the effects of antisense RNAs on various target sites in a model mRNA were examined. The target sites of antisense RNAs suppressing gene expression were identified, not only in the translation initiation region (TIR) of mRNA, but also at the junction between the coding region and 3'' untranslated region. Surprisingly, an antisense RNA recognizing the upstream region of TIR enhanced gene expression through increasing mRNA stability. [BMB Reports 2014; 47(11): 619-624]     

Differences in the translation efficiency and mRNA stability mediated by 5'-UTR splice variants of human SP-A1 and SP-A2 genes

Surfactant protein A (SP-A) plays an important role in host defense, modulation of inflammatory processes, and surfactant-related functions of the lung. The human SP-A (hSP-A) locus consists of two functional genes, SP-A1 and SP-A2. Several hSP-A 5'-untranslated region (UTR) splice variants for each gene have been characterized and shown to be translated in vitro and in vivo. In this report, we investigated the role of hSP-A 5'-UTR splice variants on SP-A production and molecular mechanisms involved. We used in vitro transient expression of hSP-A 5'-UTR constructs containing luciferase as the reporter gene and quantitative real-time PCR to study hSP-A 5'-UTR-mediated gene expression. We found that 1) the four (A'D', ABD, AB'D', and A'CD') 5'-UTR splice variants under study enhanced gene expression, by increasing luciferase activity from 2.5- to 19.5-fold and luciferase mRNA from 4.3- to 8.8-fold compared with the control vector that lacked hSP-A 5'-UTR; 2) all four 5'-UTR splice variants studied regulated mRNA stability. The ABD variant exhibited the lowest rate of mRNA decay compared with the other three constructs (A'D', AB'D', and A'CD'). These three constructs also exhibited significantly lower rate of mRNA decay compared with the control vector; 3) based on the indexes of translational efficiency (luciferase activity/mRNA), ABD and AB'D' exhibited higher translational efficiency compared with the control vector, whereas the translational efficiency of each A'D' and A'CD' was lower than that of the control vector. These findings indicate that the hSP-A 5'-UTR splice variants play an important role in both SP-A translation and mRNA stability.     

Unfolding of mRNA secondary structure by the bacterial translation initiation complex

Translation initiation is a key step for regulating the level of numerous proteins within the cell. In bacteria, the 30S initiation complex directly binds to the translation initiation region (TIR) of the mRNA. How the ribosomal 30S subunit assembles on highly structured TIR is not known. Using fluorescence-based experiments, we assayed 12 different mRNAs that form secondary structures with various stabilities and contain Shine-Dalgarno (SD) sequences of different strengths. A strong correlation was observed between the stability of the mRNA structure and the association and dissociation rate constants. Interestingly, in the presence of initiation factors and initiator tRNA, the association kinetics of structured mRNAs showed two distinct phases. The second phase was found to be important for unfolding structured mRNAs to form a stable 30S initiation complex. We show that unfolding of structured mRNAs requires a SD sequence, the start codon, fMet-tRNA(fMet), and the GTP bound form of initiation factor 2 bound to the 30S subunit.     

An unstructured mRNA region and a 5' hairpin represent important elements of the E. coli translation initiation signal determined by using the bacteriophage T7 gene 1 translation start site.

Gene 1 of bacteriophage T7 early region--the RNA polymerase gene--is very actively translated during the infectious cycle of this phage. A 29 base pair fragment of its ribosome binding site containing the initiation triplet, the Shine-Dalgarno sequence (S-D), 10 nucleotides (nt) upstream and 6 nt downstream of these central elements was cloned into a vector to control the expression of the mouse dihydrofolate reductase gene (dhfr). Although all essential parts of this translation initiation region (TIR) should be present, this fragment showed only very low activity. Computer analysis revealed a potentially inhibitory hairpin binding the S-D sequence into its stem base paired to vector-derived upstream sequences. Mutational alterations demonstrated that this hairpin was not responsible for the low activity. However, addition of 21 nt of the T7 gene 1 upstream sequence to the 29 base pair fragment were capable of increasing the translational efficiency by one order of magnitude. Computer analysis of this sequence, including nucleotide shuffling, revealed that it contains a highly unstructured region lacking mRNA secondary structures but with a hairpin at its 5' end, here formed solely by T7 sequences. There was not much difference in activity whether the mRNA included or lacked vector-derived sequences upstream of the hairpin. Such highly unstructured mRNA regions were found in all very efficiently expressed T7 genes without any obvious sequence homologies. The delta G values of these regions were higher, i.e. potential secondary structural elements were fewer, than in TIR of genes from E. coli. This is likely due to the fact that T7 as a lytic phage is relying for successful infection on much stronger signals which a cell cannot afford because of the indispensable balanced equilibria of its interdependent biochemical processes. When the 5' ends of efficient T7 gene mRNA are formed by the action of RNase III they generally start with an unstructured region. Efficiently expressed T7 genes within a polycistronic mRNA, however, always contain a hairpin preceding the structure free sequence. We suggest that the formation of this 5' hairpin is releasing enough energy to keep the unstructured regions free of secondary RNA structures for sufficient time to give ribosomes and factors a good chance for binding to the TIR. In addition, sequences further downstream of the start codon give rise to an additional increase in efficiency of the TIR by almost two orders of magnitude.