In vitro transcription assays have been used to study the rate of ribonucleic acid (RNA) synthesis from the Escherichia coli lactose promotor mutant lacL8UV5 contained on a 203-bp (base pair) restriction fragment. The half-life of long (63-base) RNA production from heparin-resistant RNA polymerase-promotor complexes was found to be related to the amount of oligonucleotides released during the initiation process (abortive initiation). Studies indicate that once a ternary complex between the promoter, RNA polymerase, and a newly synthesized RNA seven and nine nucleotides long is formed, abortive initiation is reduced and the rate of synthesis of long RNAs is increased. The promoter for the left inverted repeat of the transposable element Tn5 was also examined. It was observed to have a much slower rate of production of long RNAs, and it released oligonucleotides 4 times as often as the lactose promoter. The correlation between the amount of abortive initiation and the half-time of long RNA production is discussed.
3-Methylcrotonyl-CoA carboxylase (MCase; EC 6.4.1.4) and propionyl-CoA carboxylase (PCase; EC 6.4.1.3) have been obtained in highly purified form from bovine kidney mitochondria. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that each enzyme is composed of nonidentical subunits, including a smaller biotin-free subunit (M, 62,000 and 58,000 for MCase and PCase, respectively), and a larger biotin-containing subunit (M, 80,000 and 74,000 for MCase and PCase, respectively). The possibility that these subunits were derived from a single, larger precursor polypeptide via proteolysis was explored by purification and electrophoresis of each enzyme in the presence of protease inhibitors, but no evidence for proteolysis was obtained. Specific antisera directed towards each enzyme were prepared. The anti-PCase preparation was used to precipitate crossreacting PCase from a pig heart extract. Analysis of the immunoprecipitate obtained revealed a biotin-containing polypeptide (M, 78,000) and a biotin-free polypeptide (Mr 55,000), suggesting that pig heart PCase also contains nonidentical subunits analogous to those seen in the kidney mitochondrial MCase and PCase. A bipartite subunit structure may be a common feature in mammalian MCase and PCase.Deficiencies in two biotin-containing enzymes, propionyl-CoA carboxylase (PCase; EC 6.4.1.3) and 3-methylcrotonyl-CoA carboxylase (MCase; EC 6.4.1.4), are responsible for the genetically inherited diseases, propionicacidemia and 3-methylcrotonylglycinuria, respectively, in humans (see refs. 1-4 for a review). In order to determine the structural defect(s) in the dysfunctioning enzymes, basic information on the structure of the corresponding normal enzymes is needed. We have been working to obtain purified PCase and MCase from a convenient animal source for such studies. Bovine kidney mitochondria represent a rich source of MCase (unpublished data) and of PCase, and we have recently obtained highly purified preparations of both enzymes. Based on published properties of crystalline pig heart PCase (5) and in analogy with other biotin enzymes from animal tissues, including acetyl-CoA carboxylase (6)(7)(8) and pyruvate carboxylase (9), we had assumed that PCase and MCase from animal sources would each contain one type of multifunctional subunit with all three catalytic subsites fused into a single polypeptide chain (see refs. 10 and 11 for a review). We report here the unexpected finding that mitochondrial MCase and PCase both contain two different types of subunits, a structural pattern of biotin enzymes heretofore ascribed only to bacterial biotin enzymes (10-12).t The significance of this finding in relation to the evolution of biotin enzymes and to the interpretation of recent genetic studies on human PCase deficiency will be discussed.EXPERIMENTAL PROCEDURE Enzyme Purification. MCase and PCase were purified from bovine kidney mitochondria as outlined below. Mitochondria, isolated by differential centrifugation (15), were disrupted w...
Synthetic oligonucleotides were used to introduce mutations into the lacPUV5 promoter. Four mutations were obtained at positions -13, -14, and -15, with respect to the transcriptional start site. The effects of these mutations were measured in vivo and the results are discussed with respect to the consensus sequence and other promoter mutations located in this region.
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