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At least 127 records · Page 7Linked to original sources
PubMed1987

Factor-independent termination of transcription in a stretch of deoxyadenosine residues in the template DNA.

The primer transcript of plasmid ColE1 extends beyond the replication origin in either of two different modes. It does or does not form a hybrid with the template DNA. When a stretch of 20 deoxyadenosine residues is inserted into the template strand downstream of the origin, more than 90% of hybridized transcripts and about 10% of unhybridized transcripts terminate at the insert. When the number of inserted residues is reduced to ten, the corresponding values are decreased considerably, while the sites of termination are almost unchanged. A palindrome immediately before the stretch increased the efficiency of termination of unhybridized transcripts. Upon termination of hybridized transcripts, RNA polymerase detaches from the template. A structure made by a hybrid or by folded RNA may affect a property of transcription, and a stretch of deoxyadenosine residues including those beyond the actual site of termination may facilitate detachment of RNA polymerase from the template DNA.

Bacteriocin Plasmids↗
PubMed1993

Recognition of boxA antiterminator RNA by the E. coli antitermination factors NusB and ribosomal protein S10.

The boxA sequences of the E. coli ribosomal RNA (rrn) operons are sufficient to cause RNA polymerase to read through Rho-dependent transcriptional terminators. We show that a complex of the transcription antitermination factors NusB and ribosomal protein S10 interacts specifically with boxA RNA. Neither NusB nor S10 binds boxA RNA on its own, and neither NusA nor NusG affects the interaction of the NusB-S10 complex with boxA RNA. Mutations in boxA that impair its antitermination activity compromise its interaction with NusB and S10, suggesting that ribosomal protein S10 regulates the synthesis of ribosomal RNA in bacteria. RNA containing the closely related boxA sequence from the bacteriophage lambda nutR site is not stably bound by NusB and S10. This probably explains why antitermination in phage lambda depends on the phage lambda N protein and the boxB component of the nut site, in addition to boxA.

Bacterial Proteins↗
PubMed2002

Neurospora crassa supersuppressor mutants are amber codon-specific.

Neurospora crassa has 10 mapped supersuppressor (ssu) genes. In vivo studies indicate that they suppress amber (UAG) premature termination mutations but the spectrum of their functions remains to be elucidated. We examined seven ssu strains (ssu-1, -2, -3, -4, -5, -9, and -10) using cell-free translation extracts. We tested suppression by requiring it to produce firefly luciferase from a reading frame containing premature UAA, UGA, or UAG terminators. All mutants except ssu-3 suppressed UAG codons. Maximal UAG suppression ranged from 15% to 30% relative to controls containing sense codons at the corresponding position. Production from constructs containing UAA or UGA was 1-2%, similar to levels observed with all nonsense codons in wild-type and ssu-3 extracts. UAG suppression was also seen using [35S]Met to radiolabel polypeptides. Suppression enabled ribosomes to continue translation elongation as determined using the toeprint assay. tRNA from supersuppressors showed suppressor activity when added to wild-type extracts. Thus, these supersuppressors produce amber suppressor tRNA.

Amino Acid Sequence↗
PubMed1998

Rho-dependent termination within the trp t' terminator. I. Effects of rho loading and template sequence.

About one-half of the terminators of the Escherichia coli genome require transcription termination factor rho to function. Here we use the very "diffuse" trp t' terminator of E. coli to show that both template sequence and transcript secondary structure are involved in controlling the template positions and efficiencies of rho-dependent termination. Termination begins in the wild-type trp t' terminator sequence approximately 97 bps downstream of the promoter under our standard reaction conditions, and termination efficiencies for individual positions on three related templates have been determined in the form of quantitative patterns of rho-dependent RNA release. Comparison of these patterns shows that the rho-dependent termination efficiency at individual template positions depends primarily on the nucleotide sequence at and near the putative 3' end of the transcript, although these efficiencies can also be influenced by RNA sequence elements located further upstream. The amplitudes of the peaks of the RNA release patterns at specific template positions are controlled primarily by the effectiveness of the binding of the rho hexamer to the "rho loading site" of the transcript. Introduction of a stable element of secondary structure into the nascent RNA within the loading site both shifts the position of initial rho-dependent termination downstream and decreases the amplitudes of the peaks of the RNA release pattern at the corresponding sequences. These results and others are consistent with the view that rho-dependent terminators contain two essential components: (i) an upstream rho loading site on the RNA that is 70-80 nucleotide residues in length, essentially devoid of secondary structure, and which contains sufficient numbers of rC residues to activate the RNA-dependent ATPase of rho; and (ii) a downstream sequence within which termination actually occurs. In this study we use the trp t' terminator to characterize the involvement of each of these sequence components in detail in order to provide the parameters required to define a quantitative mechanistic model for the function of rho in transcript termination.

Base Composition↗
PubMed1998

Rho-dependent termination within the trp t' terminator. II. Effects of kinetic competition and rho processivity.

Continuing our quantitative analysis of rho-dependent termination at the trp t ' terminator, we here present evidence that the position of rho-dependent terminators along the template is strongly regulated by the secondary structure of the nascent RNA transcript, and that the prerequisite for establishing an effective kinetic competition between elongation and rho-dependent RNA release at a particular termination position is an upstream rho hexamer properly bound to a rho loading site on the nascent transcript. As a consequence kinetic competition regulates termination efficiency at individual positions downstream of the rho loading site, but does not control the position of the termination zone. Conditions that favor the formation of stable secondary structure on the RNA shift the initial rho-dependent termination position downstream. These results are consistent with a model that states that the rho protein requires approximately 70-80 nucleotide residues of unstructured RNA to load onto the transcript and cause termination, and that stable RNA secondary structures are effectively "looped out" to avoid interaction with rho, meaning that more RNA must be synthesized before rho-dependent termination can begin. Thus, although the rate of transcript elongation is important in determining termination efficiency at specific template positions, the process of loading of the rho hexamer onto the nascent transcript plays an overriding role in determining the template positions of rho-dependent terminators. We also show that at high salt concentrations, which have virtually no effect on the rate of transcript elongation, rho-dependent transcript termination is more directly dependent on the efficiency of rho loading, since the processivity of translocation of rho along the nascent transcript to "catch up with" the polymerase is much more limited under these conditions. A quantitative model for rho-dependent transcript termination is developed to account for all these interacting effects of rho on the efficiency of RNA release from actively transcribing elongation complexes.

Binding, Competitive↗
PubMed2003

Rare instances of Cre-mediated deletion product maintained in transgenic wheat.

Previously, we described a Cre-lox based strategy to convert a complex multi-copy integration pattern to a single-copy transgene (Srivastava et al., 1999). When a lox-containing transgenic line of wheat was crossed with a cre-expressing line, extra copies of the transgene were deleted by site-specific recombination. This process included the removal of a lox-flanked selection marker gene, bar. Three out of six F1 plants were chimeric for the resolved and the complex loci because both completely resolved and incompletely resolved patterns were found in the F2 population. From one F1 plant, 4 out of 20 F2 progeny showed not only incomplete resolution of the complex integration pattern, but also the presence of a circular loxP-bar-nos3' fragment, which we refer to as the bar circle. This bar circle was detected in subsequent generations, and was associated with the presence of both the lox transgene and the cre locus. We hypothesize that the cre gene in these bar circle plants must have undergone a genetic or epigenetic change that altered the spatial and/or temporal pattern of cre expression. Late expression might excise the DNA incompletely, and late in development. What is surprising is that the DNA is not degraded, but remains in the cells as an extra-chromosomal circular molecule.

Acetyltransferases↗
PubMed2004

Requirement of IS911 replication before integration defines a new bacterial transposition pathway.

Movement of transposable elements is often accompanied by replication to ensure their proliferation. Replication is associated with both major classes of transposition mechanisms: cut-and-paste and cointegrate formation (paste-and-copy). Cut-and-paste transposition is often activated by replication of the transposon, while in cointegrate formation replication completes integration. We describe a novel transposition mechanism used by insertion sequence IS911, which we call copy-and-paste. IS911 transposes using a circular intermediate (circle), which then integrates into a target. We demonstrate that this is derived from a branched intermediate (figure-eight) in which both ends are joined by a single-strand bridge after a first-strand transfer. In vivo labelling experiments show that the process of circle formation is replicative. The results indicate that the replication pathway not only produces circles from figure-eight but also regenerates the transposon donor plasmid. To confirm the replicative mechanism, we have also used the Escherichia coli terminators (terC) which, when bound by the Tus protein, inhibit replication forks in a polarised manner. Finally, we demonstrate that the primase DnaG is essential, implicating a host-specific replication pathway.

DNA Primase↗
PubMed1987

Physiological levels of normal tRNA(CAGGln) can effect partial suppression of amber mutations in the yeast Saccharomyces cerevisiae.

A number of ciliated protozoa are known to read the stop codons UAA and UAG as sense codons that specify glutamine during protein synthesis. In considering evolutionary mechanisms for this curious divergence from the standard genetic code, we propose the existence of progenitor tRNAs for glutamine that can weakly suppress UAA and UAG codons. It has been previously shown that multicopy plasmids that overexpress normal tRNA(CAAGln) and tRNA(CAGGln) genes from the yeast Saccharomyces cerevisiae can partially suppress a number of yeast ochre and amber mutations, respectively. In the present study we show that the tRNA(CAGGln) gene can also function as a weak amber suppressor when expressed in cells at physiological levels. This observation is consistent with a role of tRNA(CAGGln) as an evolutionary progenitor of tRNAs that strongly decode UAG codons.

Animals↗
PubMed1989

Gene Q antiterminator proteins of Escherichia coli phages 82 and lambda suppress pausing by RNA polymerase at a rho-dependent terminator and at other sites.

The Q genes of phages lambda and 82 encode transcription antiterminators that are active in vitro in a purified transcription system. Transcription termination is thought to involve two distinct steps: pausing of the transcription complex at the terminator and release of enzyme and RNA; either or both steps might be inhibited by Q protein. We show that Q-modified RNA polymerase pauses much less efficiently than does unmodified enzyme at the natural pause sites of a rho-dependent terminator as well as at other pause sites. This changed behavior can account for the termination properties of Q-modified RNA polymerase and reflects a fundamental alteration of the elongation properties of the enzyme.

Bacteriophage lambda↗
PubMed1989

Echinomycin-induced hypersensitivity to osmium tetroxide of DNA fragments incapable of forming Hoogsteen base pairs.

To show conclusively that the critical structural deformation of double-helical DNA that is induced by the binding of quinoxaline antibiotics does not involve the formation of Hoogsteen base pairs, we have prepared a DNA fragment containing the nucleoside analog 7-deaza-2'-deoxyadenosine in one of the two strands. This DNA fragment was subjected to treatment with the thymidine-specific reagent osmium tetroxide and to DNase I "footprinting" in the presence or absence of micromolar concentrations of echinomycin. We report that this anti-tumor antibiotic binds to DNA containing the nucleoside analog as well as to natural DNA and that the previously reported hypersensitivity to osmium tetroxide of certain thymidine residues adjacent to echinomycin binding sites is maintained in analog-containing DNA. Since these thymidines are rendered incapable of participating in Hoogsteen base pairs by the incorporation of 7-deaza-2'-deoxyadenosine, we conclude that this unusual base-pairing scheme is not the cause of the observed hypersensitivity to osmium tetroxide and that it therefore results from a large local unwinding of the DNA in the presence of the antibiotic. Moreover, preventing the possibility of Hoogsteen base pairing does not preclude echinomycin binding.

Aminoglycosides↗
PubMed1990

Sequence-specific and polarized replication termination in vitro: complementation of extracts of tus- Escherichia coli by purified Ter protein and analysis of termination intermediates.

We have developed an in vitro replication system in which purified replication termination protein (Ter) elicits specific and polarized termination of DNA replication at terminator sites (tau) in a cell extract of tus- Escherichia coli that does not encode Ter protein. Using this system and two-dimensional agarose gel electrophoresis, we have identified intermediates with stalled replication forks. The replication bubbles contain both arrested leading strands and lagging strands that were initiated at the ColE1 origin of replication and that had progressed unidirectionally until arrested at tau. To dissect the system further, we have analyzed the kinetics of the formation of the termination intermediates and have discovered that the earliest termination intermediate had properties consistent with an arrested D loop. The D loop contained an arrested leading strand. Thus, in this test system, there appears to be a transient uncoupling of leading- and lagging-strand synthesis during termination of replication at tau sites.

DNA Replication↗
PubMed1996

Bacteriophage lambda N protein alone can induce transcription antitermination in vitro.

Specific and processive antitermination by bacteriophage lambda N protein in vivo and in vitro requires the participation of a large number of Escherichia coli proteins (Nus factors), as well as an RNA hairpin (boxB) within the nut site of the nascent transcript. In this study we show that efficient, though nonprocessive, antitermination can be induced by large concentrations of N alone, even in the absence of a nut site. By adding back individual components of the system, we also show that N with nut+ nascent RNA is much more effective in antitermination than is N alone. This effect is abolished if N is competed away from the nut+ RNA by adding, in trans, an excess of boxB RNA. The addition of NusA makes antitermination by the N-nut+ complex yet more effective. This NusA-dependent increase in antitermination is lost when delta nut transcripts are used. These results suggest the formation of a specific boxB RNA-N-NusA complex within the transcription complex. By assuming an equilibrium model, we estimate a binding constant of 5 x 10(6) M-1 for the interaction of N alone with the transcription complex. This value can be used to estimate a characteristic dissociation time of N from the complex that is comparable to the dwell time of the complex at an average template position, thus explaining the nonprocessivity of the antitermination effect induced by N alone. On this basis, the effective dissociation rate of N should be approximately 1000-fold slower from the minimally processive (100-600 bp) N-NusA-nut+ transcription complex and approximately 10(5)-fold slower from the maximally processive (thousands of base pairs) complex containing all of the components of the in vivo N-dependent antitermination system.

Bacterial Proteins↗
PubMed1998

Overexpression of the SUP45 gene encoding a Sup35p-binding protein inhibits the induction of the de novo appearance of the [PSI+] prion.

[PSI+], a non-Mendelian element found in some strains of Saccharomyces cerevisiae, is presumed to be the manifestation of a self-propagating prion conformation of eRF3 (Sup35p). Translation termination factor eRF3 enhances the activity of release factor eRF1 (Sup45p). As predicted by the prion model, overproduction of Sup35p induces the de novo appearance of [PSI+]. However, another non-Mendelian determinant, [PIN+], is required for this induction. We now show that SUP45 overexpression inhibits the induction of [PSI+] by Sup35p overproduction in [PIN+] strains, but has no effect on the propagation of [PSI+] or on the [PIN] status of the cells. We also show that SUP45 overexpression counteracts the growth inhibition usually associated with overexpression of SUP35 in [PSI+] strains. We argue that excess Sup45p inhibits [PSI+] seed formation. Because Sup45p complexes with Sup35p, we hypothesize that excess Sup45p may sequester Sup35p, thereby reducing the opportunity for Sup35p conformational flips and/or self-interactions leading to prion formation. This in vivo yeast result is reminiscent of the in vitro finding by investigators of Alzheimer disease that apolipoprotein E inhibits amyloid nucleation, but does not reduce seeded growth of amyloid.

Fungal Proteins↗
PubMed1998

Escherichia coli rho factor induces release of yeast RNA polymerase II but not polymerase I or III.

Purified RNA polymerase II (pol II) from the yeast Saccharomyces cerevisiae pauses without releasing at many locations during in vitro transcription. Pausing can be induced by intrinsic DNA sequence as well as by specific DNA bound proteins such as the RNA pol I termination factor, Reb1p, or lac repressor. Addition of rho termination factor from E. coli induces RNA pol II to release at all of these pause sites. Rho-induced release of pol II requires both a rho binding site in the transcript upstream of the pause sites as well as hydrolysis of ATP. In contrast, rho factor has no effect on either pausing or release by RNA pol I or III. When combined with previous observations, these results suggest that RNA pol II may terminate by a mechanism closely related to the rho-dependent mechanism of prokaryotes. In contrast, pol I and III appear to utilize a mechanism more related to the rho-independent terminators of prokaryotes.

Adenosine Triphosphate↗
PubMed1996

Factor-dependent release of nascent RNA by ternary complexes of vaccinia RNA polymerase.

Factor-dependent transcription termination during synthesis of vaccinia early mRNAs occurs at heterogeneous sites downstream of a UUUUUNU signal in the nascent transcript. The choice of termination site is flexible and is determined by a kinetic balance between nascent chain elongation and the transmission of the RNA signal to the polymerase. To eliminate ongoing elongation as a variable, we have established a system to study transcript release by purified ternary complexes halted at a defined template position 50-nucleotides 3' of the first U residue of the termination signal. Release of the nascent RNA depends on the vaccinia termination factor (VTF) and an ATP cofactor. Transcript release is blocked by BrUMP substitution within the termination signal of the nascent RNA. In these respects, the release reaction faithfully mimics the properties of the termination event. We demonstrate that ternary complexes are refractory to VTF-mediated transcript release when the first U of the UUUUUNU signal is situated 20 nucleotides from the growing point of the nascent chain. Ribonuclease footprinting of the arrested ternary complexes defines a nascent RNA binding site on the polymerase elongation complex that encompasses a 16-21 nucleotide RNA segment extending proximally from the 3' end of the chain. We surmise that access of VTF to the signal sequence is prevented when UUUUUNU is bound within the nascent RNA binding site. Hence, physical not kinetic constraints determine the minimal distance between the signal and potential sites of 3' end formation.

Base Sequence↗
PubMed2003

Site-specific footprinting reveals differences in the translocation status of HIV-1 reverse transcriptase. Implications for polymerase translocation and drug resistance.

Resistance to nucleoside analogue inhibitors of the reverse transcriptase of the HIV-1 often involves phosphorolytic excision of the incorporated chain terminator. Previous crystallographic and modeling studies suggested that this reaction could only occur when the enzyme resides in a pre-translocational stage. Here we studied mechanisms of polymerase translocation using novel site-specific footprinting techniques. Classical footprinting approaches, based on the detection of protected nucleic acid residues, are not sensitive enough to visualize subtle structural differences at single nucleotide resolution. Thus, we developed chemical footprinting techniques that give rise to hyperreactive cleavage on the template strand mediated through specific contacts with the enzyme. Two specific cuts served as markers that defined the position of the polymerase and RNase H domain, respectively. We show that the presence of the next correct dNTP, following the incorporated chain terminator, caused a shift in the position of the two cuts a single nucleotide further downstream. The footprints point to monotonic sliding motions and provide compelling evidence for the existence of an equilibrium between pre- and post-translocational stages. Our data show that enzyme translocation is reversible and uncoupled from nucleotide incorporation and the release of pyrophosphate. This translocational equilibrium ensures access to the pre-translocational stage after incorporation of the chain terminator. The efficiency of excision correlates with an increase in the population of complexes that exist in the pre-translocational stage, and we show that the latter configuration is preferred with an enzyme that contains mutations associated with resistance to nucleoside analogue inhibitors.

Amino Acid Substitution↗