E. coli RNA polymerase

Nebularine. Nebularine(44) is a naturaHy occurring purine riboside isolated from S.jokosukanensis (1,3,4). It is phosphorylated, and inhibits purine biosynthesis and RNA synthesis, but is not incorporated into RNA by E. coli RNA polymerase. It has also found appHcation as a transition state analogue for treatment of schistosomiasis and as a substrate for the restriction endonuclease, Hindll (138—141). 

They did find that these compounds behaved kinetically as competitive inhibitors of polymerization of the normal substrates e.g., guanosine 5 -diphosphate. These authors suggested that the successful completion of the polynucleotide phosphorylase reaction requires that the nucleotide be capable of assuming the anti conformation. Also, Kapuler and Reich (53) have found that both 8-bromo- and 8-oxoguanosine 5 -triphosphates are very poor substrates in the E. coli RNA polymerase reaction and are competitive inhibitors with respect to guanosine 5 -triphosphate as a substrate. 

Finally, the pyrimidine nucleotide 4-thiouridine 5 -triphosphate, also presumably essentially in the syn conformation, cannot serve as a substrate for homopolymerization with E. coli RNA polymerase (54). Small amounts of 4-thiouridine were reported to have been found in naturally occurring transfer RNA from coli (55). Scheit has reported that as a monomer in CDCI3, the base can still pair with adenosine (56). 

Figure 5.5. A. Schematic illustration of the E. coli RNA polymerase showing the domain structure of the a subunit. The cx-NTD domain is responsible for assembly of RNAP while the a-CTD domain binds DNA and is a target for transcriptional activators. B. The two-hybrid system is based on the interaction of proteins that are fused to the X repressor and NTD domain of the a subunit of RNAP. In the example shown, Gal4 interacts with Gall IP to recruit RNAP to the promoter and activate transcription of the lacZ reporter gene. Figure adapted from Dove and Hochschild (1998).

E. coli RNA polymerase or Avian myetoblastosis DNA polymerase RNA or DNA synthesis NA + Hoffman and Niyogi 1977 Sirover and Loeb 1976... 

The sequence neutrality for random stretches of double-stranded DNA makes uranyl ion a very useful reagent for examining contact regions in protein-DNA complexes. Such photo-footprinting studies have been carried out with the A-repressor/ORl [185], E. Coli RNA polymerase/deo Plpromoter [187] and transcription factor IIIA-ICR [188]. 

RapA RapA E. coli RNA polymerase core or RNA polymerase holoenzyme Recycling of RNA polymerase [332]. 

R. J. Davenport, G. J. Wuite, R. Landick, and C. Bustamante, Single-molecule smdy of transcriptional pausing and arrest by E. coli RNA polymerase. Science 287, 2497-2500 (2000). 

To enable RNA polymerase to synthesize an RNA strand complementary to one of the DNA strands, the DNA duplex must unwind over a short distance, forming a transcription bubble. During transcription, the E. coli RNA polymerase generally keeps about 17 bp unwound. The 8 bp RNA-DNA hybrid occurs in this unwound region. Elongation of a transcript by E. coli RNA polymerase proceeds at a rate of 50 to 90 nucleotides/s. Because DNA is a helix, movement of a transcription bubble requires considerable strand rotation of the nucleic acid molecules. DNA strand rotation is restricted... 

FIGURE 26-4 Structure of the RNA polymerase holoenzyme of the bacterium Thermus aquaticus. (Derived from PDB ID 1 IW7.)The overall structure of this enzyme is very similar to that of the E. coli RNA polymerase no DNA or RNA is shown here. The j3 subunit is in gray, the j3 subunit is white the two a subunits are different shades of red the to subunit is yellow the cr subunit is orange. The image on the left is oriented as in Figure 26-6. When the structure is rotated 180° about the y axis (right) the small to subunit is visible. 

RNA Polymerase (a) How long would it take for the E. coli RNA polymerase to synthesize the primary transcript for the E. coli genes encoding the enzymes for lactose metabolism (the 5,300 bp lac operon, considered in Chapter 28) (b) How far along the DNA would the transcription bubble formed by RNA polymerase move in 10 seconds ... 

In addition, they assist in replication of RNA viruses, another process that requires RNA synthesis. Viruses sometimes make use of host RNA polymerases but often synthesize their own catalytic subunits. Bacteriophage T4 uses the E. coli RNA polymerase and o factors but modifies their action through the binding of several phage-encoded proteins.248 In contrast, phage T7 encodes its own relatively simple RNAP whose initiation complex (Section A,2)29 and elongation complexes have been studied 249-249b... 

JennerTJ, Fulford J, O Neill P (2001) Contribution of base lesions to radiation-induced clustered DNA damage implication for models of radiation response. Radiat Res 156 590-593 Jeppesen C, Buchardt O, Flenriksen U, Nielsen PE (1988) Photocleavage of DNA photofootprinting of E. coli RNA polymerase bound to promoter DNA by azido-9-acridinylamines. Nucleic Acids Res 16 5755-5770... 

Transcription by E. coli RNA polymerase occurs in three phases initiation, elongation and termination. Initiation involves binding of the enzyme to a promoter upstream of the gene. During elongation, the antisense DNA strand is used as the template so that the RNA made has the same base sequence as the sense (coding) strand, except that U replaces T. A termination signal is eventually encountered that halts synthesis and causes release of the completed RNA. 

Each of the three RNA polymerases contains 12 or more subunits, some of which are similar to those of E. coli RNA polymerase. However, four to seven subunits in each enzyme are unique to that enzyme. 

Each of the three eukaryotic RNA polymerases contains 12 or more subunits and so these are large complex enzymes. The genes encoding some of the subunits of each eukaryotic enzyme show DNA sequence similarities to genes encoding subunits of the core enzyme (a2PP ) of E. coli RNA polymerase (see Topic G2). However, four to seven other subunits of each eukaryotic RNA polymerase are unique in that they show no similarity either with bacterial RNA polymerase subunits or with the subunits of other eukaryotic RNA polymerases. 

Bahr, W., Stcnder, W., Scheit, H.-M., Jovin, T. M. Binding of rifampicin to E. coli RNA polymerase. Thermodynamic and kinetic studies. In RNA Polymerase. Cold Spring Harbor Laboratory 1976, p. 369... 

Under certain conditions, the stress-70 proteins can participate in the renaturation of denatured or inactivated proteins. The renaturation capabilities of E. coli dnaK protein have been most extensively documented. It has been shown that in vitro, dnaK can protect E. coli RNA polymerase from aggregation when the polymerase is incubated at elevated temperatures that would normally result in loss of activity, and, further, that dnaK can disaggregate and reactivate polymerase, once it has been inactivated by heat denaturation (Skowyra et al., 1990). These activities are absolutely dependent on ATP hydrolysis. The mutant dnaK756 protein is effective in protecting active RNA polymerase against heat inactivation, but is incapable of disaggregating and reactivating polymerase, once it has been heat inactivated. 

Siebenlist, U., Simpson, R.B., Gilbert, W. (1980) E. coli RNA Polymerase Interacts Homologously with Two Different Promotors, Cell 20, 269-276. 

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... 

Azidophenacyl)thioJuridine 5 -triphosphate (134) has been prepared by alkylation of 5-thlouridine 5 -triphosphate with 4-azidophenacyl bromide. This analogue is a substrate for E.coli RNA polymerase and once incorporated into RNA can be used to study RNA-protein or RNA-nucleic acids interactions by photo-activated cross-linking. Unlike other photoaffinity labels the cross-linking... 

What is the main role of the s (sigma) factor in the E. coli RNA polymerase complex ... 

There are many commercially available E. coli cell strains for the heterologous expression of recombinant proteins. By far the most common promoter system is the T7 expression system and the most commonly used expression host is BL21(DE3). If one is using a promoter that is recognized by the E. coli RNA polymerase, any cell strain can be used. Frequently useful are cell strains that supplement rare codons and strains that alter the ability of E. coli to produce disulfide bonds in the cytoplasm. However, there are cell strains that have been developed for their ability to express toxic proteins149,150 and a wide range of other strains that may be useful for any particular protein. 

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