RNA polymerase from E. coli

Protein molecules that have only one chain are called monomeric proteins. But a fairly large number of proteins have a quaternary structure, which consists of several identical polypeptide chains (subunits) that associate into a multimeric molecule in a specific way. These subunits can function either independently of each other or cooperatively so that the function of one subunit is dependent on the functional state of other subunits. Other protein molecules are assembled from several different subunits with different functions for example, RNA polymerase from E. coli contains five different polypeptide chains. 

Two aldehydic nucleotide derivatives have found use as affinity labels. The magnesium salt of (64), formed by oxidation of ATP with periodate, is a competitive inhibitor of pyruvate carboxylase with respect to [Mg. ATP2-],100 and (65), obtained from the / -anomer of 5-formyluridine-5 -triphosphate on treatment with alkali, is a non-competitive and reversible inhibitor of DNA-dependent RNA polymerase from E. coli.101 In each case, addition of borohydride gives stoicheiometric covalent linkage of the nucleotide to the enzyme, with irreversible inactivation. It is thought that condensation with lysine occurs to give a Schiff s base intermediate, which undergoes subsequent reduction. 

In thermolysin (Matthews et al., 1974 Holmes and Matthews, 1982) zinc is bound approximately tetrahedrally to glutamate (monodentate), two histidines, and water. While zinc in the native enzyme is tetracoord-inate, in some inhibitor complexes it is pentacoordinate. The four calcium ions are bound by six to eight oxygen ligands, as shown in Fig. 35, with Ca 0 distances of 2.23-2.71 A. The RNA polymerase from E. coli contains Zn(II) and Mg(II), which may be substituted by Mn(II) (Chuk-nyhVy et al., 1990). 

Rifampicin was first shown by Hartmann et al. 54 to have a specific inhibitory effect on RNA polymerase from E. coli. Later, other active ansamycins were found and RNA polymerases from a large variety of bacteria other than E. coli proved to be sensitive to the drug. More recently, an RNA polymerase from E. coli containing only one subunit and probably involved in the initiation of DNA replication (dna G gene product) has been shown to be resistant to rifampicin5 s This holds true also for the various mammalian RNA polymerases. In contrast to non-specific inhibitors of transcription such as actinomycin and mitomycin, rifampicin interacts specifically with the bacterial enzyme itself. With the aid of 14C-labelled rifampicin it could be shown that the drug forms a very stable complex with the enzyme in a molar ratio of 1 1S6> 57 The dissociation constant of this complex is 10-9 M at 37 °C and... 

Key polymerases. Compare DNA polymerase I and RNA polymerase from E. coli in regard to each of the following features (a) activated precursors, (b) direction of chain elongation, (c) conservation of the template, and (d) need for a primer. 

We begin our consideration of transcription by examining the process in bacteria such as E. coli. RNA polymerase from E. coli is a very large (-400 kd) and complex enzyme consisting of four kinds of subunits (Table 28.1). The subunit... 

Chloro-9-cyclopentyl-8-azapurine inhibited synthesis of DNA, RNA, and protein in E. coli. Blockage of thymine-nucleotide formation was the first effect seen. Alkylation of enzymes by the 6-chloro substituent was suggested as a mechanism. This azapurine inhibited the RNA polymerase from E. coli, but not that from M. lysodeikticus. Formyltetrahydrofolate synthetases, of both mammalian and bacterial origins, were strongly inhibited. The same azapurine, at 0.3 mM, markedly inhibited the steroid-induced synthesis of A -3-ketosteroid isomerase in Pseudomonas testoster-oni ... 

In contrast to the single-subunit RNA polymerase found in bacteriophages, the model prokaryotic RNA polymerase from E. coli is a multisubunit enzyme. This polymerase has a five-subunit core that forms a constricted, tunnel-shaped catalytic site [97]. Prokaryotic RNA polymerases require an additional subunit, a, for promoter-specific initiation of transcription [98, 99]. 

Recall List three important properties of RNA polymerase from E. coli. 

There has been much research on the location of the zinc ions, the interaction of template or substrate with the enzymes, and the relationship of zinc and an activator metal ion (usually magnesium) to the fidelity of transcription. The DNA-dependent RNA polymerase from E. coli which contains two moles of zinc ions per mole of enzyme has the subunit structure (X2PP o. The separated subunits obtained in the presence of 7M urea do not contain zinc. However, both the P and P subunits take up zinc ions to give 0.6 0.3 and 1.4 0.5 moles of tightly bound zinc ions per mole of subunit respectively. It was suggested that at least one of the two tightly bound zinc ions in the RNA polymerase is located in the P subunit, whilst the other Zn ion may be in P, or P, or at the contact domain of these two subunits. 

ThioUTP, or poly[d(4-thio T)1 RNA polymerase from E. coli Photoinactivation and labeling of fi and subunits 122... 

A similar mechanistic study on DNA-dependent RNA polymerase from E. coli using phosphorothioate analogues has been carried out by the group of F. Eckstein (27). 

Recently, Leibovitch and Khesin (1974) obtained single male X chromosomes which absorbed twice as much H -RNA polymerase from E. coli as did each female... 

TABLE 5. Ability of Different Reconstituted (Thymus) Nucleo-histones to Maintain RNA Synthesis by the Action of RNA-Polymerase from E. coli (Huang et al., 1964). 

Replication of synthetic homopolyribonucleotides has been studied by Friedemann Schneider and coworkers (Schneider et al., 1979 Heinrichs and Schneider, 1980). They studied RNA-synthesis at poly(A)-poly(U) templates by the unspecific RNA polymerase from E. coli in a stirred flow reactor. In general this reaction follows an overall autokatalytic kinetics. In the flow reactor the recycling process is replaced by an influx of A and an outflux of the material in the reactor. It is worth noticing that critical slowing down has been observed experimentally in this system. 

Fig. X-1. Inhibition of RNA synthesis by clupcine components and basic homopolypeptides. — The RNA synthesizing system contains a total volume of 0.5 ml aqueous solution. Reagents are added in the following order 20 pmoles of Tris-HCl, pH 8.1 6 pmoles of /5-mercaptoethanol 2 gmoles of MgCb 0.5 pmole of MnCb 190 mfxmoles of DNA, clupeine or basic homopolypeptides as indicated in the figure 100 m/imoles each of C-UTP, cold ATP, GTP and CTP and about 20 (xg of RNA polymerase from E. coli B. After 10 min incubation at 37 °C, acid-insoluble material is collected and the radioactivity...

Rabussay, D., Zillig, W. A rifampicin resistant RNA-polymerase from E. coli altered in the j5-subunit. FEBS Letters 5, 104-106 (1969). 

ZiLLiG, W., Zechel, K., Rabussay, D., Schachner, M., Sethi, V. S., Palm, P., Heil, A., Seifert, W. On the role of different subunits of DNA-dependent RNA polymerase from E. coli in the transcription process. Cold Spr. Harb. Symp. quant. Biol. 35, 47-58 (1970). 

Several laboratories have reported that the DNA-dependent RNA polymerase from E. coli can use RNA as a template under certain experimental conditions (Robertson, 1971 Fox et al., 1964 Nakamoto and Weiss, 1962 Krakow and Ochoa, 1963). 

Koch, G., Vollertsen, I. RNA-Dependent RNA polymerase from E. coli. 8th FEBS Meeting, August 20-25, 1972 (c) - Abstract. 

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