Influence of Salts on RNA Synthesis by DNA‐Dependent RNA‐Polymerase from Escherichia coli

Abstract

The effect of salt concentration on RNA synthesis by DNA-dependent RNA-polymerase from Escherichia coli has been investigated. For a double stranded template the following influences are observed: (1) Free enzyme and enzyme bound to DNA, but not synthesizing RNA, are inhibited above an ionic strength of about 0.15. (2) The rate of RNA-synthesis, by those enzyme molecules which have started RNA chains, is stimulated by increasing the salt concentration up to an optimum. Magnesium salts cause a larger stimulation than monovalent cations. However, the optimal effect always occurs at an ionic strength of about 0.36. (3) This stimulatory effect is distinguished from the cofactor function which requires only low concentrations of divalent cations (Mg2+, Co2+, Mn2+). (4) RNA synthesis stops at low ionic strength (< 0.2) due to a first order inactivation of the synthesizing enzyme. A t high ionic strength synthesis continues over many hours a t a slowly decreasing rate. Raising the ionic strength after RNA synthesis has stopped at low ionic strength causes an immediate resumption of synthesis. Mg2+ and spermidine are most effective in this action. However, Co2+ and Mn2+ are unable to reactivate the enzyme once it has stopped.With single stranded DNA as template neither a first order inactivation of synthesizing enzyme a t low ionic strength nor a stimulation of RNA synthesis by monovalent> salts is observed. The effect of divalent cations on enzyme activity can be due to their cofactor function.The DNA-dependent RNA-polymerase from Escherichia coli is strongly influenced by changes in the ionic strength. At low ionic strength (between 0.02 and 0.12) it has been shown to exist in a form sedimenting a t 24s [l-51; a t high ionic strength the enzyme reversibly dissociates into subunits of 13s [2,4-61. At low salt concentration the polymerase forms a complex with DNA [1,7--12j which by raising the ionic strength is dissociated into free DNA and the i3S form of the enzyme [Z, 13-15].The polymerization process itself has been reported to be inhibited only after some time of synthesis at high ionic strength [5,13]. However, the effect of ionic strength on this phase of the reaction has not been extensively investigated.This communication presents a detailed analysis of the effect of ionic strength on the kinetics of RNAsynthesis with double and single-stranded DNA. It is shown that increasing the ionic strength causes Non-Standard Abbreviations. PPO, 2,5 diphenyloxazol; POPOP, 1,4-bis-2-(4 methyl4 phenoxazoly1)-bcnzene; TMA, buffer, containing: 0.01 M Tris acetate, 0.022 M NH,CI, 0.01 M magnesium acetate, 1 mM 2-mercaptoethanol, pH 7.3; TCA, trichloroacetic acid.Enzymes. DNA-dependent RNA-polymerase or nucleosidetripliosphate : RNA nucleotidyltransferase (EC 2.7.7.6) ; Ribonuclease I (EC 2.7.7.17); Pyruvate kinase (EC 2.7.1.40). a striking stimulation of both the rate and extent of RNA synthesis. This eliminates the typical plateau in the kinetics [l6-211 and allows RNA-synthesis t o continue over many hours. Moreover, synthes...

Transcription of Mammalian Chromatin by Mammalian DNA‐Dependent RNA Polymerases

“…This finding is in disagreement with that of Spelsberg and Hnilica [13] who found that as much as 2Z0/, of histone f l was released from chromatin by 0.20 M Thus we would predict that the RNA-polymerase molecules which are bound to the chromatin and are actively involved in RNA synthesis at the time of the isolation of the chromatin are unable to continue transcription until protein which is blocking further movement of the polymerase along the DNA is released from the template. At high salt concentration therefore, the RNA-polymerase activity is probably only due to the elongation of previously initiated RNA chains as the elongation process is not (in bacterial systems anyway) sensitive to high ionic strength [51]. This process will be discussed further below.…”

“…by Rifampicin APIO-13 Three steps in RNA synthesis in vitro can be distinguished each of which have certain ionic strength requirements : (a) the binding of the enzyme to the DNA and the stabilisation of the complex by the first nucleotide in the RNA chain (initiation) which can take place up to an ionic strength of about 0.4 [51,52]; (b) elongation of the RNA chain which appears to be independent of the ionic strength up to quite high levels [51]; (c) release and reinitiation of new RNA chains which only occurs a t an ionic strength of about 0.2 (see review [24]). This data has all been derived from the study of the DNA-dependent RNA polymerase of Escherichia coli.…”

Transcription in vitro of bacteriophage lambda 4S RNA: studies on termination and rho protein

“…This RNA, which is transcribed from a point near the origin of X replication (20,21), provides an attractive system for several reasons. First, the nucleotide sequence of the 4S RNA as well as the sequence of Xpga18 for approximately 35 nucleotides beyond the 3' end of the transcript have been determined (22). Second, the similarities between 4S RNA and an RNA "leader" sequence in the tryptophan operon suggest that in vivo the 4S may serve as a leader and its termination site as an attenuator (23,24).…”

Net synthesis of short RNA chains by E. coli RNA polymerase at low pH

“…The size of RNA synthesized in vitro under these conditions with T3, T7 or T4 DNA as template is of 3000 to 7000 nucleotides length [8,9]. Only at relatively high ionic strength under these pH conditions is the enzyme able to reinitiate [10]. However at low pH (5.7-6.0) and low ionic strength (3-12 mM Mg 2+ and 10-40 mM KC1), the rate of RNA synthesis was much lower, but did not reach a plateau and continued for several hours with a rate of synthesis much lower than at optimal conditions ( fig.la).…”