Preinitiation Complex Formation Research Articles

Formation of stable preinitiation complexes between eukaryotic class B transcription factors and promoter sequences.

At least one of the HeLa cell factors necessary for correct in vitro transcription from conalbumin or adenovirus late promoters binds to the region of the template that contains the ‘TATA’ box. This binding, which occurs in the absence of RNA polymerase B, results in the formation of stable preinitiation complexes.

Formation of stable preinitiation complexes is a prerequisite for ribosomal DNA transcription in vitro.

Cytoplasmic extracts from cultured mouse cells contain the factor(s) required for specific transcription initiation of rDNA by RNA polymerase I. Prior to transcription the essential proteins bind to the ribosomal gene and remain bound to the template for several rounds of transcription. The assembly of these preinitiation complexes in vitro has been demonstrated by kinetic analysis of the transcription reaction and by competition experiments. Complex formation involves an initial, rapid binding of transcription factor(s) to rDNA sequences followed by additional events which arrange the DNA-protein complex into a transcriptionally active state. Once the complexes have formed they persist for at least 2 hours in vitro and are resistant to elevated salt concentrations. The assembly of the complexes was inhibited when the template DNA was incubated with histones prior to the addition of S-100 extract. If, however, preinitiation complex formation was allowed to occur before the addition of histones, the interference of histones with specific transcription was much less pronounced.

36] Assay of eukaryotic initiation factor eIF-2 activity with extracts of interferon-treated cells

Publisher Summary This chapter presents that incubation of interferon-treated cell extracts with dsRNA leads to a reduction in their capacity to initiate protein synthesis. Thus, the incorporation of [ 35 S]methionine in the NH 2 -terminal positions of peptides synthesized in vitro is analyzed by a modification of the Edman degradation procedure and found to be reduced by dsRNA. Furthermore, the incorporation of [ 35 S]methionine into a peak sedimenting at approximately 40 S is measured by sedimentation on sucrose density gradients and also shown to be sensitive to dsRNA. The parallel activation of an endonuclease activity by dsRNA should not affect, the measurement of initiation complex formation. Since other steps in the protein synthetic pathway could be rate limiting, and it is attempted to compare the relative rates of initiation by direct measurement of the production of NH2-terminal methionine residues and have utilized both preinitiation complex formation and the Edman degradation procedure to give an overall view of the initiation process. The Edman procedure, however, has certain drawbacks. Activation of endonuclease activity causes degradation of mRNA, which probably results in fewer intact 5' termini being available for initiation; this probably explains the limited ability of initiation factors to reverse the inhibition.

Effect of chemical modification of supercoiled simian virus 40 DNA on the rate of in vitro transcription

Superhelical simian virus 40 FI DNA could be modified with the single-strand-specific reagent N-cyclohexyl-N'-beta-(4-methylmorpholinium)ethylcarbodiimide (CMC). A limited reaction, of less than 2% of the base pairs, resulted in almost total inhibition of in vitro transcription by DNA-dependent RNA polymerase from Escherichia coli. This effect was shown to be due to DNA modification and not to inhibition of polymerase activity by the reagent. Inhibition of enzyme activity occurred if the contaminating reagent was not absorbed with another protein before polymerase addition. No inhibition was observed when DNA and polymerase were incubated together to allow the formation of pre-initiation complexes before CMC was added. Studies of template saturation with polymerase showed that the inhibition of transcription by DNA modification was due to a loss of binding ability of the enzyme to the reacted, supercoiled DNA when reaction times of less than 2 h were used.

Effect of salt on the transcription of T7 DNA by RNA polymerase from T4 phage-infected E.coli

Transcription of T7 DNA by T4 core enzyme with host sigma is more sensitive to KCI than that by host core enzyme with host sigma. When salt is added after initiation of RNA chains has occurred, it is not inhibitory. Salt affects the binding of T4 enzyme to T7 DNA to the same degree as the binding of host enzyme. Active preinitiation complex formation is inhibited more by salt with the T4 enzyme and the inhibition is temperature-dependent.

Effect of estrogen on gene expression in the chick oviduct. Studies on the initiation of RNA synthesis on chromatin in vitro.

Studies on the effect of the temperature requirement for RNA chain initiation by Escherichia coli RNA polymerase on DNA, chromatin, and reconstituted chromatin were carried out in order to better understand the nature of the initiation process. Varying the temperature or ionic strength during preincubations had little effect on the formation of stable preinitiation complexes (RS complexes) between RNA polymerase and chromatin. This observation was in marked ocntrast to similar studies performed on native DNA and indicates that initiation sites for RNA synthesis on chromatin are different from those on double-stranded DNA and resemble more closely initiation of RNA synthesis on single-stranded DNA. These results suggest that the local unwinding of the initiation region which is required for RNA chain initiation on native DNA may not be a prerequisite for RNA initiation on chromatin. The results of studies on reconstituted chromatin devoid of different classes of chromatin proteins demonstrate that both histone and nonhistone fractions are essential in maintaining the characteristics inherent to initiation of RNA synthesis on chromatin. Removal of either moderatley lysine-rich histone or arginine-rich histone fractions led to the complete loss of the characteristic "chromatin type" initiation pattern for RNA synthesis, whereas, removing lysine-rich (Fi) histone had no effect. Single strand specific and double strand specific antibodies to DNA and a single strand specific nuclease were used as probes to examine the structure of chromatin sites for initiation of RNA synthesis. Their effects on RNA synthesis suggest that initiation sites on chromatin are not located in freely accessible single-stranded or double-stranded regions of DNA.

Selective effects of MgCl2 and temperature on the initiation of transcription at lac, gal, and lambda promoters.

We have studied the effect of Mg2+ on the formation of transcription preinitiation complexes (open complexes) at two adenosine 3':5'-monophosphate (cyclic AMP)-cyclic AMP receptor protein (CRP)-dependent promoters (lac and gal) and two phage lambda promoters, PL and PR. Mg2+ strongly interferes with open complex formation at the lac and gal promoters, partially inhibits the lambda PR promoter, and is without effect on the lambda PL promoter. Mutations in the lac and gal promoters can affect the response of the promoter to Mg2+. Cyclic AMP and CRP specifically reduce the inhibition of the lac and gal promoters by Mg2+. These factors also affect open complexes at lac and gal by (a) increasing the rate at which they are formed and (b) lowering the midpoint of the temperature transition curve for their formation by about 10 degrees. Open complexes at the lac promoter are more unstable to cooling, even in the presence of CRP and cyclic AMP, than open complexes at lambda promoters. Our studies suggest that the DNA of the lac and gal promoter regions is more resistant to denaturation than the DNA of phage promoter regions. Cyclic AMP and CRP act to decrease this stability, stimulating open complex formation under conditions unfavorable for DNA melting, e.g. low temperature and high Mg2+ concentrations.

Temperature and salt effects on the formation of preinitiation complexes between RNA polymerase and phage DNA

The influence of temperature and KCl concentration on the formation of rifampicin-resistant preinitiation complexes by holo RNA polymerase has been compared for T4 DNA and Azotobacter phage A21 DNA. The sharp transition with respect to temperature between an inactive complex of polymerase and DNA and a preinitiation complex reflects an equilibrium between the two complexes, the position of which depends on the temperature and the salt concentration. The transition is shifted to higher temperatures by increasing the KCl concentration. The position of this transition is characteristically different for T4 and A21 DNA. The midpoint for A21 DNA is about 15°C above that for T4 at 0.006 M KCl. At 0.15 M KCl the transition for A21 DNA cannot be observed below 37°C. This difference is responsible for the apparent inhibition of A21 DNA transcription by KCl and for the low template activity of A21 DNA under the conditions of the standard assay. Both holo and core RNA polymerases are able to form complexes with A21 DNA that are resistant to attack by rifampicin. The second-order rate constant for the inactivation of the complex with the core enzyme is three times greater than that for the complex with the holoenzyme.

Inhibition of DNA-dependent rna polymerase of E.coli by phospholipids

Summary DNA-dependent RNA polymerase of E. coli is strongly inhibited by phosphatidylglycerol and cardiolipin at concentrations of 5 to 8 × 10−5 M. The two lipids inhibit the formation of rifampicin-resistant preinitiation complexes. Phosphatidylethanolamine is not inhibitory, and at a tenfold higher concentration partially overcomes the inhibition by the other two lipids.

Activation of Transcription at Specific Promoters by Glycerol

Abstract Glycerol added to a transcription system containing λgal DNA and Escherichia coli RNA polymerase holoenzyme stimulates total RNA synthesis and replaces the requirement for cyclic adenosine 3' : 5'-monophosphate (cAMP) and cAMP receptor (CRP) for promotion of gal RNA synthesis. The stimulatory effect which is proportional to the concentration of glycerol up to 20% (v/v) occurs at the level of preinitiation complex formation. In stimulating gal transcription, glycerol appears to act at or close to the same site as cAMP and CRP because: (a) glycerol has little effect on gal transcription at saturating levels of cAMP and CRP; (b) although glycerol stimulates transcription from λgal DNA bearing revertible gal promoter mutations, transcription from DNA containing a gal promoter deletion is not stimulated; (c) glycerol stimulation of gal promoter mutants is inhibited by cAMP and CRP; and (d) with either glycerol or cAMP and CRP, transcription of the promoter-proximal galE region precedes transcription of the galKT region. The effect of glycerol on λ early transcription is to stimulate early r-strand RNA synthesis but not early l-strand RNA synthesis. However, if the DNA template contains a defective λsex promoter, the decreased levels of early l-strand RNA are restored to normal by glycerol. Ethylene glycol, dimethylsulfoxide, sucrose, and 1,3-propanediol, all of which lower the Tm of DNA, also stimulate both total RNA and gal RNA synthesis. Furthermore, the glycerol-promoted formation of the gal preinitiation complex is strongly dependent on the preincubation temperature. At lower temperatures, complex formation requires increased glycerol or decreased KCl concentrations. These findings suggest that glycerol may act by melting or changing the conformation of the DNA promoter regions.

Role of Cyclic Adenosine 3′,5′-Monophosphate and the Cyclic Adenosine 3′,5′-Monophosphate Receptor Protein in the Initiation of lac Transcription

Abstract Optimal transcription of the lac operon in a defined purified in vitro system is dependent upon the concentrations of cyclic adenosine 3',5'-monophosphate (cyclic AMP), the cyclic AMP-binding protein (CRP) and RNA polymerase. The concentration of CRP required for maximal lac transcription varies proportionately with the concentration of lac DNA used as template. Maximal lac transcription is obtained with less CRP when lac ps DNA is used as template than with wild type lac DNA. Both in vivo and in vitro the lac ps mutation appears to result in a more efficient lac promoter. The formation of a preinitiation complex which will synthesize lac mRNA upon addition of nucleoside triphosphates requires preincubation of λh80dlac DNA, RNA polymerase, CRP, and cyclic AMP. The formation of this preinitiation complex is both time and temperature dependent. A lag period of about 2 min is observed between the binding of RNA polymerase to lac DNA and the appearance of detectable lac specific hybridizable RNA. This lag period is possibly due to a slow transition occurring between the formation of the lac preinitiation complex and the onset of extensive polymerization. Our experiments provide further evidence that CRP and cyclic AMP act on lac DNA to stimulate an early step in the initiation of lac transcription by RNA polymerase.

A thermostable protein factor acting on in vitro DNA transcription

A heat-stable protein factor has been purified from E. coli supernatant fractions which has the property to greatly stimulate in vitro DNA transcription when RNA polymerase is limiting. The step in transcription which is influenced by this factor is the formation of preinitiation complexes.