Noguchi T, Yamada K, Inoue H, Matsuda T, Tanaka T
Department of Nutrition and Physiological Chemistry, Osaka University Medical School, Japan.
J Biol Chem. 1987 Oct 15;262(29):14366-71.
cDNA clones for rat R-type pyruvate kinase and a genomic clone encoding both L- and R-type isozyme mRNAs were isolated. Their sequences were compared with that of the L-type isozyme cDNA to determine the sequences of mRNA and protein of the R-type isozyme and the organizations of the L- and R-type genes. Results showed that the R-type isozyme mRNA had an identical nucleotide sequence to that of the L-type except in the 5'-terminal region including the coding sequence and the length of the 3'-untranslated region. The sequence upstream of the 5th coding residue of the L-type was replaced by a 98-nucleotide coding sequence plus a 5'-untranslated region in the R-type isozyme. Therefore, the R-type subunit consists of 574 amino acids, which is 31 residues longer than the L-type at the amino terminus. The pyruvate kinase L gene is present as a single copy per haploid genome and is composed of 12 exons and 11 introns with a length of about 9.3 kilobase pairs. The first (exon R) and second (exon L) exons encode the 5'-terminal sequences specific for the R- and L-types, respectively. The remaining downstream exons encode a sequence common to both isozymes. The last exon contains the entire 3'-untranslated region, including several putative polyadenylation signals. Alternative use of these signals is reported to be responsible for generation of multiple mRNA species for the L-type, whereas the R-type uses only the first signal. The cap site is mapped 16 nucleotides upstream from the translation initiation site for the L-type, whereas multiple cap sites were suggested for the R-type. The canonical promoter of the TATA box was identified in the upstream sequence of exon L, but not in that of exon R. Instead, the 5'-flanking region of exon R contained another promoter sequence of the CAT box. Thus, we conclude that the L- and R-type isozymes of pyruvate kinase are produced from a single gene by use of different promoters.
分离出了大鼠R型丙酮酸激酶的cDNA克隆以及一个编码L型和R型同工酶mRNA的基因组克隆。将它们的序列与L型同工酶cDNA的序列进行比较，以确定R型同工酶的mRNA和蛋白质序列以及L型和R型基因的结构。结果表明，R型同工酶mRNA除了在包括编码序列的5'-末端区域和3'-非翻译区的长度外，其核苷酸序列与L型相同。L型第5个编码残基上游的序列被R型同工酶中的一个98个核苷酸的编码序列加上一个5'-非翻译区所取代。因此，R型亚基由574个氨基酸组成，在氨基末端比L型长31个残基。丙酮酸激酶L基因在单倍体基因组中以单拷贝形式存在，由12个外显子和11个内含子组成，长度约为9.3千碱基对。第一个（外显子R）和第二个（外显子L）外显子分别编码R型和L型特异的5'-末端序列。其余下游外显子编码两种同工酶共有的序列。最后一个外显子包含整个3'-非翻译区，包括几个假定的聚腺苷酸化信号。据报道，这些信号的交替使用导致了L型产生多种mRNA种类，而R型只使用第一个信号。帽位点在L型翻译起始位点上游16个核苷酸处定位，而R型则提示有多个帽位点。在外显子L的上游序列中鉴定出了TATA盒的典型启动子，但在外显子R的上游序列中没有。相反，外显子R的5'-侧翼区域包含CAT盒的另一个启动子序列。因此，我们得出结论，丙酮酸激酶的L型和R型同工酶是通过使用不同的启动子从单个基因产生的。
