Modification Of RNA Polymerase Research Articles

Role of the spacer boxA of Escherichia coli ribosomal RNA operons in efficient 23 S rRNA synthesis in vivo

A boxA sequence, known to be important for transcriptional antitermination, is found in both the leader region and in the spacer between the 16S and 23S genes of Escherichia coli ribosomal RNA operons. We have shown that a functional leader boxA is important for efficient completion of 16 S rRNA transcription. In this study, point mutations were introduced into the 16S-23S spacer boxA of a plasmid-encoded E. coli rrnB operon in order to study the contribution of this conserved sequence element to ribosomal RNA synthesis in vivo. The rrnB mutant constructs contained an additional point mutation in each of the 16S and 23S genes, which were used to distinguish rRNA derived from plasmid and chromosomal rrn operons by primer extension analysis. Mutations in the spacer boxA reduced the proportion of plasmid-derived 23 S rRNA without affecting synthesis of plasmid-derived 16 S rRNA or spacer boxA RNA, indicating that premature termination of transcription occurred during 23 S rRNA synthesis. Reductions in plasmid-derived 23 S rRNA were very similar for total cellular RNA, 50 S subunits and 70 S ribosomes, suggesting that plasmid-derived rRNAs from mutant operons were functional in ribosome biogenesis. In the presence of a wild-type leader boxA, single nucleotide exchanges in the spacer boxA reduced the proportion of plasmid-derived 23 S rRNA from 70% to about 55% under conditions of exponential growth in rich medium. This proportion further decreased to 20 to 25% with an additional point mutation in the leader boxA. We conclude that modification of RNA polymerase into a termination-resistant form has to be renewed at the spacer boxA in order to ensure the faithful completion of full-length 23 S rRNA.

TATA-binding protein is limiting for both TATA-containing and TATA-lacking RNA polymerase III promoters in Drosophila cells.

We have investigated the role of the TATA-binding protein (TBP) in modulating RNA polymerase (Pol) III gene activity. Epitope-tagged TBP (e-TBP) was both transiently and stably transfected in Drosophila Schneider S-2 cells to increase the total cellular level of TBP. Analysis of the transcripts synthesized from cotransfected tRNA and U6 RNA genes revealed that both types of RNA Pol III promoters were substantially stimulated by an increase in e-TBP in a dose-dependent manner. Furthermore, a TBP-dependent increase in the levels of endogenous tRNA transcripts was produced in the stable line induced to express the e-TBP. We further determined whether the ability of increased TBP to induce RNA Pol III gene expression was due to a direct effect of increased TBP complexes on RNA Pol III gene promoters or an indirect consequence of enhanced expression of RNA Pol II genes. A TBP expression plasmid (e-TBP332), containing a mutation within the highly conserved carboxy-terminal domain, was both transiently and stably transfected into S-2 cells. e-TBP332 augmented the transcription from two RNA Pol II gene promoters indistinguishably from that observed when e-TBP was expressed. In contrast, e-TBP332 was completely defective in its ability to stimulate either the tRNA or U6 RNA gene promoters. In addition, increasing levels of a truncated TBP protein containing only the carboxy-terminal region failed to induce either the tRNA or U6 RNA gene promoter, whereas it retained its ability to stimulate an RNA Pol II promoter. Thus, the TBP-dependent increase in RNA Pol II gene activity is not sufficient for enhanced RNA Pol III gene transcription; rather, a direct effect on RNA Pol III promoters is required. Furthermore, these results provide the first direct evidence that the amino-terminal region of TBP is important for the formation or function of TBP-containing complexes utilized by TATA-less and TATA-containing RNA Pol III promoters. Together, these studies demonstrate that TBP is limiting for the expression of both classes of RNA Pol III promoters in Drosophila cells and implicate an important role for TBP in regulating RNA Pol III gene expression.

Control of transcription termination in prokaryotes.

A growing number of genetic systems have been shown to be controlled at the level of premature termination of transcription. Genes in this class contain transcription termination signals in the region upstream of the coding sequence. The activity of these regulatory termination signals is controlled through a variety of mechanisms. These include modification of RNA polymerase to a terminator-resistant, or terminator-prone form, and alterations in the structure of the nascent transcript, to determine whether the stem-loop structure of an intrinsic terminator or an alternate antiterminator is formed. Structural alterations in the transcript can be controlled by the kinetics of translation of the RNA, by binding of specific regulatory proteins, and by mRNA-tRNA interactions. This review describes a number of variations on the termination control theme that have been uncovered in prokaryotes.

Promoter Proximal Sequences Modulate RNA Polymerase II Elongation by a Novel Mechanism

The adenovirus major late arrest site blocks transcription by mammalian RNA polymerase II in vitro downstream of the major late promoter but not the mouse β-globin promoter. We localized the sequences responsible for anti-arrest to the 5′ end of the β-globin transcript and demonstrated that anti-arrest required that this region of RNA form base pairs with the nascent transcript upstream of the arrest site. Small antisense RNA or DNA oligonucleotides hybridizing upstream of the arrest site also prevented arrest when added in trans. Our results suggest that arrest is accompanied by retraction of the nascent transcript into the interior of the polymerase and that hybridization of the transcript prevents this movement, thereby allowing the polymerase to continue elongation.

Transcription antitermination: the lambda paradigm updated.

Coliphage lambda employs systems of transcription termination and antitermination to regulate gene expression. Early gene expression is regulated by the phage-encoded N protein working with a series of Escherichia coli proteins, Nus, at RNA sites, NUT, to modify RNA polymerase to a termination-resistant form. Expression of lambda late genes is regulated by the phage-encoded Q antitermination protein. Q, which appears to use only one host factor, acts at a DNA site, qut, to modify RNA polymerase to a termination-resistant form. This review focuses on recent studies which show that: (i) N can mediate antitermination in vitro, independent of Nus proteins. (ii) Early genes in another lambdoid phage HK022 are also regulated by antitermination, where only an RNA signal appears necessary and sufficient to create a termination-resistant RNA polymerase. (iii) A part of the qut signal appears to be read from the non-template DNA strand. (iv) A host-encoded inhibitor of N antitermination appears to act through the NUT site as well as with the alpha subunit of RNA polymerase, and is antagonized by NusB protein.

Function of a nontranscribed DNA strand site in transcription elongation

A prolonged pause in transcription elongation at positions +16 and +17 of the phage λ late gene operon has an important role in the modification of RNA polymerase by the λ gene Q transcription antiterminator. Mutations included in the transcription bubble of the paused transcription complex, particularly at +2 and +6, abolish pausing and the ability of Q protein to modify RNA polymerase. By transcribing heteroduplex templates made in vitro, we show that the sites identified by these mutations act through the nontranscribed strand of DNA. This result suggests unexpected regulatory functions of the nontranscribed DNA strand in transcription.

Characterization of a protein that interacts with the rat ribosomal gene promoter and modulates RNA polymerase I transcription

An RNA polymerase I core promoter binding factor (CPBF) was purified to apparent homogeneity from rat adenocarcinoma ascites cells by chromatographic fractionation on a series of columns including an oligodeoxynucleotide affinity column. The final preparation contained two polypeptides with molecular masses of 44,000 and 39,000 daltons. The binding of the factor to the promoter was demonstrated by Southwestern blotting, UV cross-linking and electrophoretic mobility shift assay. The specificity of its binding to the core promoter was confirmed by competitive electrophoretic mobility shift assay using several unlabeled oligo probes in the assay. The addition of increasing amounts of purified CPBF to the in vitro transcription reaction that contains a limiting quantity of the factor resulted in dramatic stimulation of RNA polymerase I (pol I) transcription of rat ribosomal RNA gene. The transcription stimulatory activity associated with the purified CPBF fractions co-purified with the core promoter binding activity in an electrophoretic mobility shift assay. Finally, in a reconstitution transcription system which is devoid of the factor and is incapable of ribosomal gene transcription, purified CPBF could trans-activate the pol I promoter. These data indicate that CPBF is a novel pol I promoter binding factor required for ribosomal gene transcription.

Enhancer 1 binding factor, a Ku-related protein, is a positive regulator of RNA polymerase I transcription initiation.

We have previously characterized a protein, enhancer 1 binding factor (E1BF), from rat cells that can modulate RNA polymerase I-directed transcription of the rat rRNA gene in vitro. E1BF, a heterodimeric DNA binding protein composed of 72-kDa and 85-kDa subunits, is related to the human Ku autoantigen with respect to immunological and certain structural properties. To establish the direct role of E1BF in transcription, we investigated the effect of anti-Ku antibodies on RNA polymerase I-directed transcription in rat and mouse cell extracts. These antibodies, one directed against the 70-kDa Ku subunit and the other against a peptide fragment of this subunit, dissociated the E1BF heterodimer into its two subunits. The DNA-protein complex formed in the presence of the antibodies contained only the 72-kDa subunit. Preincubation of the extracts with these antibodies resulted in an almost complete inhibition of transcription. The reduced transcription was observed when either linear or circular template was used. The inhibitory effect of the antibodies was greatest when added prior to preinitiation complex formation and was minimized significantly when added after establishment of the initiation complex. The repression of rRNA gene transcription was overcome by the addition of purified E1BF. This study demonstrates that E1BF, a Ku-related protein, is required for RNA polymerase I-directed transcription, the 72-kDa subunit is the major DNA binding polypeptide, the factor acts primarily in the formation of the preinitiation complex, and heterodimerization of its two subunits is crucial for maintaining the functional integrity of the protein.

Transcriptional antitermination.

Antiterminator proteins control gene expression by recognizing control signals near the promoter and preventing transcriptional termination which would otherwise occur at sites that may be a long way downstream. The N protein of bacteriophage lambda recognizes a sequence in the nascent RNA, and modifies RNA polymerase by catalysing the formation of a stable ribonucleoprotein complex on its surface, whereas the lambda Q protein recognizes a sequence in the DNA. These mechanisms of antitermination in lambda provide models for analysing antitermination in viruses such as HIV-1 and in eukaryotic genes.

Control of gene expression by lipophilic hormones

Regulation of gene expression in response to steroids, thyroid hormone, and retinoids is mediated by an impressive array of intracellular receptors. Sequence analysis showed that the hormone receptors comprise a large superfamily of ligand-responsive transcription factors. Upon binding to hormones, the receptors interact with specific hormone response elements located in the promoters of numerous genes. Promoter-bound receptors communicate with distinct receptors and/or additional members of the transcriptional machinery, frequently evoking protein-protein interactions. Ultimately, this results in the induction of complex gene systems that control hormone-induced processes such as differentiation, cell growth, and homeostasis. In addition to the genes transcribed by RNA polymerase II, the lipophilic hormones, particularly glucocorticoids, can also modulate RNA polymerase I-directed transcription of the ribosomal gene. For both transcription systems, activation and repression of genes in response to hormones have been reported. Finally, the involvement of hormone receptors in tumorigenesis has been discussed. It is likely that receptor studies will have major implications in the diagnosis and therapy of diseases such as leukemia.

Host factor requirements for processive antitermination of transcription and suppression of pausing by the N protein of bacteriophage lambda.

The N protein of phage lambda prevents termination of transcription by Escherichia coli RNA polymerase at Rho-dependent and -independent terminators in the lambda early operons. The modification of RNA polymerase by N requires an N-utilization (nut) site, present in each lambda early operon, and involves the E. coli factors NusA, NusB, NusG, and ribosomal protein S10. We show that, in the presence of NusA, N inhibits pausing by RNA polymerase and Rho-dependent termination in vitro at three sites in the lambda terminator tR1 which are located less than 100 base pairs downstream from nutR. NusA is also sufficient for partial antitermination at sites located farther downstream from nutL and nutR if there is a high concentration of N in the reaction. At low concentrations of N, the additional factors NusB, S10, and NusG are essential for antitermination at distal sites. In these conditions, the presence of NusA, NusB, S10, and NusG in the reaction enables N-modified RNA polymerase to elongate efficiently and processively through Rho-dependent and -independent terminators over distances as great as 7 kilobases downstream from the lambda nut sites. This substantial processivity of antitermination in vitro also occurs in vivo and probably reflects the stable association of N, NusA, NusB, S10, and NusG with RNA polymerase and nut site RNA in elongation complexes transcribing the lambda chromosome.

The phage λ gene Q transcription antiterminator binds DNA in the late gene promoter as it modifies RNA polymerase

The bacteriophage λ gene Q transcription antiterminator modifies RNA polymerase during an extended pause in elongation at nt +16 and +17 of the phage late gene promoter transcript. We show here that Q binds a specific DNA site between the −10 and −35 elements of the promoter as it interacts with the enzyme. We show that the pause must reflect a specialized elongation structure that is receptive to modification by Q, because Q does not bind to RNA polymerase stopped artificially after transcribing 16 nt of mutant DNA that does not encode the natural pause. Footprinting shows that RNA polymerase in the paused complex makes distinctive interactions with DNA in the region where Q binds; binding of Q, in turn, changes the footprint both at the Q-binding site and in the transcription bubble. Binding of Q to the paused transcription complex is stabilized by the transcription factor NusA, as expected from the dependence of λ Q-mediated anti-termination on NusA.

Purified MotA protein binds the -30 region of a bacteriophage T4 middle-mode promoter and activates transcription in vitro.

The bacteriophage T4-encoded MotA protein is critical for transcription from T4 middle-mode promoters. However, a direct interaction of this protein with a middle-mode promoter has not previously been demonstrated. We have cloned the motA gene and overexpressed the gene product using the T7 expression system. A simple procedure was then developed to purify the MotA protein to homogeneity. Using the purified protein we have demonstrated that MotA protein binds to the -30 region of the middle-mode promoter PuvsY. This promoter has previously been shown to be a necessary component of a T4 replication origin, and thus MotA is also a T4 origin-binding protein. Modified RNA polymerase purified from T4-infected cells was used to establish middle-mode transcription in vitro. Transcription from PuvsY was markedly enhanced by the addition of MotA protein, whether or not the template contained the cytosine modifications characteristic of T4 DNA. However, transcription from PuvsY was apparently independent of the MotA protein when unmodified RNA polymerase from uninfected cells was used.

Growth phase-dependent modification of RNA polymerase in Escherichia coli

During the transition of Escherichia coli cultures from exponential growth to stationary phase, the pre-existing RNA polymerase was found to be converted into at least three different holoenzyme forms, which could be separated by phosphocellulose column chromatography. The relative levels of these three holoenzyme forms changed depending on the phase of cell growth. The altered stationary phase forms of RNA polymerase showed promoter recognition properties that were different from those of the holoenzyme from exponentially growing cells. Enzyme reconstitution experiments showed that the altered promoter selectivity was due to modification of the core enzyme. We propose that modulation of RNA polymerase plays a role in the global switch of gene expression during the transition from exponential growth to stationary phase.

Gene product dsbA of bacteriophage T4 binds to late promoters and enhances late transcription

Gene product 33 of phage T4 is known to be essential in late transcription. Upstream from gene 33 and overlapping its 5' terminal sequence by 20 bp, we identified an open reading frame coding for a binding protein for double-stranded DNA (DsbA). Gene product DsbA is composed of 89 amino acid residues with a Mr of 10376 kDa. We purified this protein to homogeneity from over-expressing cells. Gel retardation assays reveal that it binds to DNA and footprint analyses disclose that it interacts preferentially with T4 late promoter regions. At the sites of binding the protein introduces nicks in double-stranded DNA. In vitro transcription assays performed with T4 late modified RNA polymerase on restriction fragments harbouring a T4 late promoter region prove that gene product DsbA enhances transcription from these promoter regions in the presence of gene product 33. Gene dsbA is distinct from gene das which maps close to this genomic region.

Specific modification of Escherichia coli RNA polymerase with monomercury derivative of fluorescein acetate

The method of specific modification of RNA polymerase with a monomercuric fluoresceine derivative, fluorescein-monomercuriacetate (FMMA), is proposed. Under an appropriate condition of modification, FMMA is capable of mercaptid bonding with one of the α-subunits. It is shown that covalent modification with FMMA does not affect the kinetic parameters ( K D and k 2) of RNA synthesis nor does it lead to the inhibition of the overall RNA synthesis. The spectral characteristics of FMMA covalently bound to RNA polymerase were found to be sensitive to some temperature-induced conformational alterations of RNA polymerase, indicating that the labeled enzyme allows study of conformational behaviour of RNA polymerase during its functioning.

Characterization of the late-gene regulatory region of phage 21

A segment of Escherichia coli bacteriophage 21 DNA encoding the late-gene regulator, Q21, and the late-gene leader RNA segment was sequenced; its structure is similar to those of the related phages lambda and 82. The leader RNA is about 45 nucleotides long and consists essentially entirely of sequences encoding the p-independent terminator that is the putative target of the antitermination activity of Q21. Like the corresponding regions of lambda and 82, the 21 late-gene promoter segment encodes an early transcription pause in vitro, at about nucleotide 18, during which Q21 presumably acts to modify RNA polymerase. The 21 Q gene, cloned in isolation, is active on the late-gene leader segment in trans, and its purified product is active as an antiterminator in vitro; Q21 represents a third late-gene antiterminator, in addition to those of lambda and 82. There is little evident similarity in the primary sequences of the three Q genes.

Initiation and regulation mechanisms of ribosomal RNA transcription in the eukaryote Acanthamoeba castellanii

Acanthamoeba rRNA transcription involves the binding of a transcription initiation factor (TIF) to the core promoter of rDNA to form the preinitiation complex. This complex is formed in the absence of RNA polymerase I, and persists for multiple rounds of initiation. Polymerase I next binds to form the initiation complex. This binding is DNA sequence-independent, and is directed by protein-protein contacts with TIF. DNA melting occurs in a separate step. In contrast to most prokaryotic transcription, melting occurs only following nucleotide addition and beta-gamma hydrolysis of ATP is not required as for polymerase II. Growth-dependent regulation of rRNA transcription is accomplished by modification of RNA polymerase I. The inactive form of polymerase (PolE) is unable to bind to the promoter and has altered heat stability. PolE is still active in elongation; thus, the modification affects the polymerase site involved in TIF contact. Modification of a polymerases I and III common subunit has been detected leading to the suggestion that transcription of stable RNAs of the ribosome might be co-regulated by this mechanism.

Inhibition of transcription of cytosine-containing DNA in vitro by the alc gene product of bacteriophage T4

The alc gene product (gpalc) of bacteriophage T4 inhibits the transcription of cytosine-containing DNA in vivo. We examined its effect on transcription in vitro by comparing RNA polymerase isolated from Escherichia coli infected with either wild-type T4D+ or alc mutants. A 50 to 60% decline in RNA polymerase activity, measured on phage T7 DNA, was observed by 1 min after infection with either T4D+ or alc mutants; this did not occur when the infecting phage lacked gpalt. In the case of the T4D+ strain but not alc mutants, this was followed by a further decrease. By 5 min after infection the activity of alc mutants was 1.5 to 2.5 times greater than that of the wild type on various cytosine-containing DNA templates, whereas there was little or no difference in activity on T4 HMdC-DNA, in agreement with the in vivo specificity. Effects on transcript initiation and elongation were distinguished by using a T7 phage DNA template. Rifampin challenge, end-labeling with [gamma-32P]ATP, and selective initiation with a dinucleotide all indicate that the decreased in vitro activity of the wild-type polymerase relative to that of the alc mutants was due to inhibition of elongation, not to any difference in initiation rates. Wild-type (but not mutated) gpalc copurified with RNA polymerase on heparin agarose but not in subsequent steps. Immunoprecipitation of modified RNA polymerase also indicated that gpalc was not tightly bound to RNA polymerase intracellularly. It thus appears likely that gpalc inhibits transcript elongation on cytosine-containing DNA by interacting with actively transcribing core polymerase as a complex with the enzyme and cytosine-rich stretches of the template.

Specificity and mechanism of antitermination by Q proteins of bacteriophages λ and 82

Lambdoid phage late gene operons are positively regulated by genome-specific antiterminator proteins encoded by the Q gene of each phage. In this paper, we compare the activity of phage λ and phage 82 Q proteins. Q82-mediated antitermination, like that of Qλ, involves a transcription pause during which the regulator can modify RNA polymerase. We show that the activities of both Q82 and Qλ are genome-specific in chasing RNA polymerase out of the early pause sites and in mediating antitermination. Finally, we show that the length of the RNA in the paused complex, or the exact position of the pause, affects the efficiency with which Q82 chases RNA polymerase out of the pause.