Characterization of human UMP-CMP kinase enzymatic activity and 5' untranslated region.

We have cloned a cDNA for human UMP-CMP kinase from a macrophage cDNA library. Sequence analysis showed that this cDNA is derived from the same gene as a previously reported EST-derived cDNA. Here we show that a conspicuous difference between these two clones, 73 additional 5' nucleotides in the EST clone, including a putative translational start site, is not functionally significant. This work shows that the additional 5'sequence in the EST clone was unnecessary for enzymatic activity and nonfunctional in the initiation of translation. Specifically, we found that protein expressed by both the macrophage-derived cDNA and the extended cDNA had the same relative molecular mass, consistent with use of an ATG internal to the macrophage-derived clone as the functional start site. In addition, this work more precisely defines the catalytic activity of UMP-CMP kinase. Here, we show a 3-fold greater substrate preference for CMP relative to UMP, identify ATP and UTP as the preferred phosphate donors for the reaction, and demonstrate that the reaction is Mg2+-dependent. In addition, investigation of UMP-CMP-kinase expression revealed two mRNA products in immune tissues and cancer cell lines. The smaller RNA product was previously undescribed.     

Statistical analysis of the 5' untranslated region of human mRNA using "Oligo-Capped" cDNA libraries

We constructed 34 types of human "full-length enriched" and "5'-end enriched" cDNA libraries based on the "Oligo-Capping" method. We randomly picked and sequenced 10,000 clones from these libraries. BLAST analysis showed that about 50% of the cDNAs were identical to known genes. Among them, we selected 954 species of cDNA that should represent the entire sequence from the mRNA start sites. Compared with previously reported sequences, they were on average 45 bp longer in the 5'-end. Using these cDNA data, we statistically analyzed the sequence features of the 5'UTR. The average length of the 5'UTR was 125 bp, and there was little correlation with the corresponding mRNA length (correlation coefficient = 0.26). Of the 954 species of 5'UTR, 459 contained no in-frame terminator codon, which is against the common belief. Two hundred seventy-eight species contained at least one ATG codon upstream of the initiator ATG codon. We identified 569 upstream ATGs, in total, 63% of which adequately satisfied Kozak's criteria. These findings are contrary to the typical translation initiation model, which states that translation is initiated from the "first" ATG codon.     

Increased synthesis of human parathyroid hormone in Escherichia coli through alterations of the 5' untranslated region

The expression of human parathyroid hormone (hPTH) in Escherichia coli was optimized by variations of the spacing sequence between the ribosome-binding site (RBS) and the beginning of the gene (ATG) and by increasing the complementarity of the RBS to the 16 S rRNA. The expression level of 3 micrograms/liter increased more than 100-fold to 475 micrograms/liter as a direct consequence of modifications in the region 5' of the gene.     

3′端非翻译区对重组人肝细胞生成素表达不均一性的影响

在工程菌pBV－hHPO／DH5α中表达的人肝细胞生成素（hHPO）以两种分子形式存在,经分析可能是由于表态质粒中hHPO基因的终止密码子TAG的通读造成的。表达载体经改构后,去掉质粒子hHPO基因终止密码子后的非翻译区,采用TAA作终止密码子,结果改构后的表达载体只表达单一形式的hHPO。     

G+C-rich tract in 5' end of human introns.

Analysis of an artificial neural network trained to classify DNA as coding or non-coding revealed compositional differences between sequence parts translated into protein and those that were not. The 5' end of human introns was found to have a base composition that was non-random to an extent matching the non-randomness in the 3' end that contains the polypyrimidine tract. The prevailing nucleotides in the initial 50 nucleotides of human introns are guanine and cytosine, the trinucleotide GGG was found to occur almost four times as frequently as it would in sequences with a uniform distribution of the nucleotides. The initial part of terminal exons and their associated terminal introns were shown to have a very special base composition deviating strongly from the normal picture in other exons and introns.     

Characterization of protein-binding to the spinach chloroplast psbA mRNA 5' untranslated region.

RNA-binding proteins play a major role in regulating mRNA metabolism in chloroplasts. In this work we characterized two proteins, of 43 and 47 kDa, which bind to the spinach psbA mRNA 5' untranslated region (psbA encoding the D1 protein of photosystem II). The 43 kDa protein, which is present in the stroma and in membranes, co-sediments with a complex of 68S. It was purified, and the N-terminal sequence was determined. Upon homology search it was identified as the chloroplast homologue of the Escherichia coli ribosomal protein S1. The 47 kDa protein, which, in contrast with the 43 kDa protein, sediments with a small sedimentation coefficient, is only detected in the stromal fraction. It is soluble in an uncomplexed form. By deletion analysis, an element within the psbA mRNA 5' untranslated region was identified that is necessary but not sufficient for binding of stromal proteins. The 'central protein binding element' ranges from nucleotide -49 to -9 of the psbA mRNA 5' untranslated region. It comprises the Shine-Dalgarno-like GGAG motif and, 7 nucleotides upstream, an endonucleolytic cleavage site involved in psbA mRNA degradation in vitro . The mechanistic impacts of this region in relation to RNA-binding proteins are discussed.