Mutations affecting translation of the bacteriophage T4 rIIB gene cloned in Escherichia coli

Summary Mutant ribosome binding sites of the bacteriophage T4 rIIB gene, resident on an 873 bp DNA fragment, were cloned into a plasmid vector as in-frame fusions to a reporter gene, beta-galactosidase. The collection of mutations included changes in the region 5 to the Shine/Dalgarno sequence, a mutation of the Shine/Dalgarno sequence, the alternate initiation codons GUG, AUA and ACG, and mutants in which several closely spaced initiation codons compete with each other on the same mRNA. The results show that the secondary structure variations we have installed 5 to the Shine/Dalgarno sequence have little effect on translation. GUG is essentially as good an initiator of translation as AUG when they are assayed on separate messages, but is outcompeted at least 50-fold in the sequence AUGUG. AUA and ACG are poor start codons, and are temperature sensitive. The initiation codon pair AUGAUA, in which the AUG is only two nucleotides from the Shine/Dalgarno sequence, displays a novel cold-sensitive phenotype.     

Influence of mRNA determinants on translation initiation in Escherichia coli.

We have studied the classic initiation elements of mRNA sequence and structure to better understand their influence on translation initiation rates in Escherichia coli. Changes introduced in the initiation codon, the Shine and Dalgarno sequence, the spacing between those two elements, and in the secondary structures within initiation domains each change the rate of 30 S ternary complex formation. We measured these differences using extension inhibition analysis, a technique we have called "toeprinting". The rate of 30 S initiation complex formation in the absence of initiation factors agrees well with in vivo translation rates in some instances, although in others a regulatory role of initiation factors in 30 S complex formation is likely. Nucleotides 5' to the Shine and Dalgarno domain facilitate ternary complex formation.     

Influence of the spacer region between the Shine–Dalgarno box and the start codon for fine-tuning of the translation efficiency in Escherichia coli

Translation efficiency contributes several orders of magnitude difference in the overall yield of exogenous gene expression in bacteria. In diverse bacteria, the translation initiation site, whose sequence is the primary determinant of the translation performance, is comprised of the start codon and the Shine–Dalgarno box located upstream. Here, we have examined how the sequence of a spacer between these main components of the translation initiation site contributes to the yield of synthesized protein. We have created a library of reporter constructs with the randomized spacer region, performed fluorescently activated cell sorting and applied next-generation sequencing analysis (the FlowSeq protocol). As a result, we have identified sequence motifs for the spacer region between the Shine–Dalgarno box and AUG start codon that may modulate the translation efficiency in a 100-fold range.     

Use of the 'Perceptron' algorithm to distinguish translational initiation sites in E. coli.

We have used a "Perceptron" algorithm to find a weighting function which distinguishes E. coli translational initiation sites from all other sites in a library of over 78,000 nucleotides of mRNA sequence. The "Perceptron" examined sequences as linear representations. The "Perceptron" is more successful at finding gene beginnings than our previous searches using "rules" (see previous paper). We note that the weighting function can find translational initiation sites within sequences that were not included in the training set.     

The construction of a synthetic Escherichia coli trp promoter and its use in the expression of a synthetic interferon gene.

An 82 base pair DNA fragment has been synthesised which contains the E. coli trp promoter and operator sequences and also encodes the first Shine Dalgarno sequence of the trp operon. This DNA fragment is flanked by EcoRI and ClaI/TaqI cohesive ends and is thus easy to clone, transfer between vector systems and couple to genes to drive their expression. It has been cloned into plasmid pAT153, producing a convenient trp promoter vector. We have also joined the fragment to a synthetic IFN-alpha 1 gene, using synthetic oligonucleotides to generate a completely natural, highly efficient bacterial translation initiation signal on the promoter proximal side of the IFN gene. Plasmids carrying this construction enable E. coli cells to express IFN-alpha 1 almost constitutively and with significantly higher efficiency than from a lacUV5 promoter based system.     

Secondary structure as primary determinant of the efficiency of ribosomal binding sites in Escherichia coli.

Using a previously described vector (pKL203) we fused several heterologous ribosomal binding sites (RBSs) to the lacZ gene of E. coli and then studied the variation in expression of the fusions. The RBSs originated from bacteriophage Q beta and MS2 genes and the E. coli genes for elongation factor EF-Tu A and B and ribosomal protein L11 (rplK). The synthesis of the lacZ fusion proteins was measured by an immuno precipitation method and found to vary at least 100-fold. Lac-specific mRNA synthesis follows the variation in protein production. It appears that there is a correlation between the efficiency of an RBS to function in the expression of the fused gene and the lack of secondary structure, involving the Shine and Dalgarno nucleotides (SDnts) and/or the initiation codon. This efficiency is context dependent. The sequence of the SD nts and the length and sequence of the spacer region up to the initiation codon alone are not able to explain our results. Deletion mutations, created in the phage Q beta replicase RBS, reveal a complex pattern of control of expression, probably involving the use of a "false" initiation site.     

Translational reinitiation in the presence and absence of a Shine and Dalgarno sequence.

The process of translational reinitiation in Escherichia coli was studied in a two cistron system where expression of the downstream reporter gene was dependent on translation of an upstream reading frame. The dependence was almost absolute. Upstream translation increased expression of the downstream gene by two to three orders of magnitude. This large difference allowed us to quantitate restarts in a meaningful manner. In the absence of a Shine and Dalgarno (SD) region reinitiation occurred but its efficiency was about 10% of that found in the SD carrying counterpart. We discuss three ways by which translational coupling between neighboring cistrons can be enforced.     

Comparison of mRNA features affecting translation initiation and reinitiation

Regulation of gene expression at the level of translation accounts for up to three orders of magnitude in its efficiency. We systematically compared the impact of several mRNA features on translation initiation at the first gene in an operon with those for the second gene. Experiments were done in a system with internal control based on dual cerulean and red (CER/RFP) fluorescent proteins. We demonstrated significant differences in the efficiency of Shine Dalgarno sequences acting at the leading gene and at the following genes in an operon. The majority of frequent intercistronic arrangements possess medium SD dependence, medium dependence on the preceding cistron translation and efficient stimulation by A/U-rich sequences. The second cistron starting immediately after preceding cistron stop codon displays unusually high dependence on the SD sequence.     

Quantitative analysis of ribosome binding sites in E.coli.

185 clones with randomized ribosome binding sites, from position -11 to 0 preceding the coding region of beta-galactosidase, were selected and sequenced. The translational yield of each clone was determined; they varied by more than 3000-fold. Multiple linear regression analysis was used to determine the contribution to translation initiation activity of each base at each position. Features known to be important for translation initiation, such as the initiation codon, the Shine/Dalgarno sequence, the identity of the base at position -3 and the occurrence of alternative ATGs, are all found to be important quantitatively for activity. No other features are found to be of general significance, although the effects of secondary structure can be seen as outliers. A comparison to a large number of natural E.coli translation initiation sites shows the information profile to be qualitatively similar although differing quantitatively. This is probably due to the selection for good translation initiation sites in the natural set compared to the low average activity of the randomized set.     

Proteins encoded by the DpnII restriction gene cassette. Two methylases and an endonuclease

Proteins encoded by three genes in the DpnII restriction enzyme cassette of Streptococcus pneumoniae were purified and characterized. Large amounts of the proteins were produced by subcloning the cassette in an Escherichia coli expression system. All three proteins appear to be dimers composed of identical polypeptide subunits. One is the DpnII endonuclease, and the other two are DNA adenine methylase active at 5' GATC 3' sites. Inactivation of enzyme activity by insertions into the genes and comparison of the DNA sequence with the amino-terminal sequence of amino acid residues in the proteins demonstrated the following correspondence between genes and enzymes. The promoter-proximal gene in the operon, dpnM, encodes a 33 X 10(3) Mr polypeptide that gives rise to a potent DNA methylase. The next gene, dpnA, encodes the 31 x 10(3) Mr polypeptide of a weaker and less-specific methylase. The third gene, dpnB, encodes the 34 x 10(3) Mr polypeptide of the endonuclease. Although the endonuclease polypeptide is initiated from an ordinary ribosome-binding site, each of the methylase polypeptide begins at an atypical site with a consensus sequence entirely different from that of Shine & Dalgarno. This presumptive novel ribosome-binding site is well recognized in both S. pneumoniae and E. coli.     

油菜叶绿体核糖体蛋白基因rps7的克隆和序列分析(英文)

从甘蓝型油菜 (Brassicanapuscv .H1 65)叶绿体基因组克隆得到了编码核糖体蛋白的基因rps7。经序列分析得知 ,该基因编码区包含 468个核苷酸 ,编码一个分子量为 2 0 1 0 9D、由 1 55个氨基酸组成的蛋白质。该基因的核苷酸和编码的氨基酸序列与烟草对应基因的同源性皆高达 97% ;而与水稻对应基因的同源性分别为 90 %和 84%。该基因不含内含子 ,没有典型的SD序列 ,但在 5’端 - 2 5～- 2 2位发现一个与烟草psbA基因的顺式作用元件RBS2完全相同的TGAT框。与烟草和水稻的同源序列比较 ,该基因在 3’端非编码区变异较大 ,发生了多次插入和缺失。构建了包含该基因在内的一个 1 .0kbDNA的限制性内切酶图谱。所报告的基因序列已登录GenBank。     

Bicistronic expression strategy for high‐level expression of recombinant proteins in Corynebacterium glutamicum

Directly using the promoter associated with 5′‐untranslated region of a high‐protein‐abundance gene from the genome may cause low expression activity of an expression system. A bicistronic expression part containing the short 5′ coding sequence of the source gene and an embedded Shine–Dalgarno sequence can cause higher expression levels of the recombinant gene in a bicistronic cassette. Here, we evaluated two methods to construct expression parts and exploited genomic sequence sources to provide specific functional sequences to complete the expression system. The architecture of the bicistronic part increased the expression levels of target genes and performed more reliably than conventional expression parts with the same promoter and 5′ untranslated region. For Corynebacterium glutamicum, the strongest bicistronic part, HP‐BEP4, was obtained from a heterologous sequence source, leading to a 2.24‐fold increase in the expression level of fluorescent protein over constitutively expressed pXMJ19 or the production of more than 100 mg/L single‐chain variable fragment (scFv). It could meet the needs of overexpressing key genes in C. glutamicum.     

The Shine and Dalgarno hypothesis for termination: the 3' terminus of the 16S rRNA of the Escherichia coli ribosome can be modified or base-paired with a complementary oligonucleotide without affecting termination in vitro

The occurrence of the nucleotides "...CCUUAOH" at the 3' terminus of the 16S rRNA of the small subunit of the Escherichia coli ribosome led to the suggestion that they may have a direct base pairing with the termination codon in the termination event of protein biosynthesis (Shine and Dalgarno 1974). We have examined this concept with two approaches, firstly using a 30S subunit whose 16S rRNA has been modified with a fluorescein moiety on the terminal adenosine together with the antibody against the moiety, and secondly with an oligonucleotide, UAAGG, complementary to the terminal pentanucleotide sequence of the rRNA. Collectively the data suggest that the nucleotides at the 3' terminus of 16S rRNA are not critically involved in base pairing during termination codon recognition.