RNA polymerase in E. coli

MECHANISM FIGURE 26-1 Transcription by RNA polymerase in E. coli. For synthesis of an RNA strand complementary to one of two DNA strands in a double helix, the DNA is transiently unwound, (a) About 17 bp are unwound at any given time. RNA polymerase and the bound transcription bubble move from left to right along the DNA as shown facilitating RNA synthesis. The DNA is unwound ahead and rewound behind as RNA is transcribed. Red arrows show the direction in which the DNA must rotate to permit this process. As the DNA is rewound, the RNA-DNA hybrid is displaced and the RNA strand extruded. The RNA polymerase is in close contact with the DNA ahead of the transcription bubble, as well as with the separated DNA strands and the RNA within and immediately behind the bubble. A channel in the protein funnels new nucleoside triphosphates (NTPs) to the polymerase active site. The polymerase footprint encompasses about 35 bp of DNA during elongation. 

Proteins in addition to p mediate and modulate termination. For example, the nusA protein enables RNA polymerase in E. coli to recognize a characteristic class of termination sites. In E. coli, specialized termination signals called attenuators are regulated to meet the nutritional needs of the cell (Section 31.4.1). A common feature ofprotein-independent and proteindependent termination is that the functioning signals lie in newly synthesized RNA rather than in the DNA template. 

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). 

The RNA polymerase of E. coli possesses with its subimit construction (a2PP o) a simple structure in comparison to eucaryotic RNA polymerases. The sigma factor is only required for the recognition of the promoter and the subsequent formation of a tight complex. After the incorporation of the first 8-10 nucleotides into the transcript, the sigma factor dissociates from the holoenzyme, and the remaining core enzyme carries out the rest of the elongation. 

Despite the complexity of the processes represented by Eq. 28-6, a yeast cell is able to transcribe genes at rates of about one in every 6-8 s.496 This can be compared with a rate of about once in 2-3 s for RNA polymerase of E. coli. 

There are two enzymes that can catalyze the synthesis of RNA with the help of a DNA template. One of these, called primase is encoded by the dnaG gene. Primase catalyzes the synthesis of small primer RNAs that are required for DNA synthesis. The other enzyme called RNA polymerase catalyzes the synthesis of all the other RNAs found in E. coli. For some time it was thought that RNA polymerase catalyzed the synthesis of the primers that initiate replication of the chromosome. Presently, it is believed that all primer synthesis is catalyzed by primase. 

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... 

Wehrli, W., Handschin, J., Wunderli, W. Interaction between rifampicin and DNA-dependent RNA polymerase of E. coli. In RNA Polymerase. Cold Spring Harbor Laboratory 1976, p. 397... 

Fig. 5. Lineweaver-Burk plot of the effect of increasing concentrations of template DNA on the incorporation of 3H-AMP into RNA by RNA polymerase of E. coli K-12, in the absence of...

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 ... 

RNA polymerase in h. coli is a multisubunit enzyme. The subunit composition of the ". SOO-kd holoenzyme is u >3 3 a and that of the core enzyme is ajpp. IVanscription is initiated at promoter sites consisting of two sequences, one centered near —10 and the other near — TS that is, 10 and d5 nucleotides away from the start site in the 5 (upstream) direction. The consensus sequence of the —10 region is TATA AT The a subunit enables the holoenzyme to recognize promoter sites. When the growth temperature is raised. E. coli expresses a special ex subunit that selectively binds the distinctive promoter of lieat-shock genes, RNA polymerase must unwind the... 

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]. 

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. 

Match the subunit of the RNA polymerase of E. coli in the left column with its putative function during catalysis from the right column. 

DNA, coding for the GFP, was introduced in liposomes composed by a phospholipid mixture (a), together with the whole T T machinery (T7 RNA polymerase and E. coli cell extracts). GFP was synthesized inside large MLVs prepared by the dehydration/ rehydration method. 

I have talked mostly about eukaryotic cells, so I should mention here the regulation of RNA polymerase of E. coli by an ADPRT of phage T4. This example, and the presence of ADPRT enzymes in protozoa such di Physarum polycephaluniy Tetrahymena pyriformis, Dictyosteleum discoideum, Trypanosoma cruzi, Trypanosoma brucei and probably va Plasmodium spp. show that ADPRT enzymes evolved at a fairly early stage in evolution. 

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