Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Rho Factor”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources
PubMed1981

Control of bacteriophage phi X 174 gene expression by the transcription termination factor rho in vitro.

The effect of rho factor on bacteriophage phi X 174 gene expression was studied in a cell-free, DNA-dependent, RNA-directed protein synthesizing system (transcription-translation coupled system) using S30 extracts of wild-type and rho-mutant Escherichia coli cells. It has been found that in the presence of a functionally active and endogenous or added rho factor, only a limited number of the viral genes (A, B, and D) are expressed effectively, whereas the expression of genes, F, G, and H, which encode capsid proteins, is strongly inhibited. Mutational or thermal inactivation of rho factor results in a considerable activation of capsid protein synthesis and, at the same time, in some depression of the synthesis of gene A, B, and D products. From the results of this study it is suggested that phage phi X 174 has two classes of gene, late and early, which are separated on the chromosome by a rho-dependent transcription terminator. We propose that the expression of these classes is regulated by a termination-antitermination mechanism.

Bacteriophage phi X 174↗
PubMed1993

Selective enrichment of RNA species for tight binding to Escherichia coli rho factor.

We have applied the SELEX procedure (systematic evolution of ligands by exponential enrichment) to obtain RNA molecules that bind tightly to the Escherichia coli transcription termination factor rho. The starting pool was a population of RNA molecules 77 nucleotides (nt) long, in which was embedded a cassette of 30 nt of randomized sequence. The apparent dissociation constant of this RNA pool for hexameric rho factor was about 1 microM. After eight rounds of selection by filter binding, with RNA in either 10-fold or 40 to 100-fold excess at each step, the dissociation constant of the selected RNA had dropped by more than 500-fold to about 1 nM. Analysis of 29 clonal isolates from the population revealed that five had KDs substantially weaker than 10 nM (presumably background carryover), 40% were C-rich (as might have been predicted from rho's known substrate binding), and 40% had a strikingly preserved potential hairpin, in most cases of 6 base pairs with a 3 nt CAA loop and preceded by a CCCCA consensus. The rho-dependent trp t' terminator region includes a related potential hairpin structure; however, it is energetically unfavorable. The implications of the sequence findings for elucidating both static and dynamic aspects of rho factor recognition and response to its RNA target site are discussed.

Base Sequence↗
PubMed1975

Transcription termination factor rho activity is altered in Escherichia coli with suA gene mutations.

Rho factor has been purified from a strain of E. coli containing the Su78 mutation in the suA gene and assayed in another strain with an amber mutation in the suA gene. The rho from the Su78 mutant strain is present in normal amounts but has altered termination function; it does not terminate transcription at some sites that are recognized effectively by the rho factor from the isogenic wild-type strain. Rho in cells with an amber mutation in the suA gene has been assayed by its RNA-dependent ATPase activity. Extracts of cells of this strain have only 9% as much of this rho activity as extracts of cells of the isogenic wild-type strain. These results suggest that rho is the product of the suA gene. Since mutations in the suA gene are known to decrease polar effects of mutations in other genes, it is also suggested that rho factor is at least partially responsible for polar effects.

Bacterial Proteins↗
PubMed1985

[Effect of mutation changes in RNA-polymerase and transcription termination factor rho on expression of various operons in E. coli].

Six mutations, impairing DNA polymerase of E. coli in combination with the wild type gene for rho factor or ts-mutation rho 15 have been studied in relation to the expression of seven operons having different types of regulation. The expression of genes for glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase is shown to be constitutive and resistant to mutationally altered RNA polymerase and rho factor. The expression of genes for adenine phosphoribosyltransferase and of deo operon is regulated by rho dependent attenuators with attenuation being lifted incomplete medium. Mutation rho 15 decreases the level of enzymes of thr and lac operons independent of mRNA levels of these operons. Mutation rho 15 effect on posttranscriptional level is modified by mutations damaging RNA polymerase. The data obtained suppose RNA polymerase to affect all stages of realization of genetic information, beginning with promoter recognition and RNA synthesis and including the protein synthesis on mRNA.

DNA Restriction Enzymes↗
PubMed1996

Characterization of an unusual Rho factor from the high G + C gram-positive bacterium Micrococcus luteus.

A transcription termination factor (Rho) was purified from the Gram-positive bacterium Micrococcus luteus, and the complete gene sequence was determined. The M. luteus Rho polypeptide has 690 residues, which is 271 residues more than its homolog from Escherichia coli. Most of the additional residues compose a highly charged, hydrophilic segment that is inserted in a non-conserved region between two conserved regions of the RNA-binding domain of the known Rho homolog proteins. This segment extends from residues 49 to 311 and includes a stretch of 238 residues that contain no hydrophobic side chains. Biochemical studies indicate that the M. luteus protein is very similar to E. coli Rho in terms of its RNA-dependent NTPase activity and its sensitivity to the Rho-specific inhibitor bicyclomycin. However, the M. luteus protein has a less stringent RNA cofactor specificity. It also acts to terminate RNA transcription with E. coli RNA polymerase on the lambda cro DNA template, but at much earlier termination stop points than those recognized by E. coli Rho. Thus, the M. luteus protein functions as a true Rho factor, but with a different specificity than that of E. coli Rho. We propose that this altered specificity is consistent with its need to function on transcripts that have a high content of G + C residues.

Amino Acid Sequence↗
PubMed1994

Evidence supporting a tethered tracking model for helicase activity of Escherichia coli Rho factor.

Transcription termination factor Rho of Escherichia coli has an ATP-dependent RNA.DNA helicase activity that presumably facilitates RNA transcript release from the elongation complex. This helicase activity is unidirectional (5' to 3') and is stoichiometric, with one RNA molecule released per Rho hexamer in vitro. A simple RNA tracking model postulates that after Rho's initial binding, it translocates preferentially toward the 3' end of the RNA. Nitrocellulose filter binding studies combined with RNase H cleavage are inconsistent with this simple tracking model. Instead, they support a model in which Rho forms tight primary binding interactions with the recognition region of the RNA and remains bound there while transient secondary RNA binding interactions coupled to ATP hydrolysis serve to scan along the RNA to contact the RNA.DNA helix. This "tethered tracking" model is consistent with other properties of Rho factor, including the presence of two classes of RNA binding sites on the Rho hexamer and the 1:1 stoichiometry in the Rho helicase assay.

Adenosine Triphosphate↗
PubMed1980

Control of early gene expression of bacteriophage T4: involvement of the host rho factor and the mot gene of the bacteriophage.

Many early mRNA species of bacteriophage T4 are not synthesized after infection of Escherichia coli in the presence of chloramphenicol. This has been interpreted as a need for T4 protein(s) to be synthesized to allow expression of some early genes, e.g., those for deoxycytidinetriphosphatase, deoxynucleosidemonophosphate kinase and UDP-glucose-DNA beta-glucosyltransferase. In the experiments described here, early mRNA of bacteriophage T4 was allowed to accumulate during chloramphenicol treatment. After the addition of rifampin to inhibit further RNA synthesis, and subsequent removal of chloramphenicol, the accumulated mRNA was permitted to express itself into measured enzyme activities. It was shown that the early mRNA species coding for deoxycytidinetriphosphatase and UDP-glucose-DNA beta-glucosyltransferase could be formed in the presence of chloramphenicol if the E. coli host cell carried a mutation in the structural gene for the RNA chain termination factor rho. This was interpreted to mean that T4 protein(s) with anti-rho activity is normally required for the expression of these two early genes. An altered rho-factor could not, however, relieve the need of phage protein synthesis for the formation of another early mRNA, that coding for deoxynucleosidemonophosphate kinase. In this case the mot gene of T4 seemed to be involved, since the primary infection of E. coli cells with the mot gene mutant tsG1 did not allow subsequent deoxynucleoside monophosphate kinase mRNA synthesis after wild-type phage infection in the presence of chloramphenicol. In control experiments, deoxynucleoside monophosphate kinase mRNA synthesis induced by wild-type phage superinfecting in the presence of chloramphenicol was facilitated by the primary infection with T4 phage containing an unmutated mot gene.

Chloramphenicol↗
PubMed1981

Bacteriophage phi X174-specific mRNA synthesis in cells deficient in termination factor rho activity.

A previous report (Hayashi et al., Proc. Natl. Acad. Sci. U.S.A. 73:3519-3523, 1976) indicated that in vivo bacteriophage phi X174 mRNA's terminate after genes J, F, G, and H. However, termination at these sites is not stringent. To determine whether termination of phi X174 transcription depends on rho factor activity, we introduced a temperature-sensitive rho mutation (nitA) into a phi X174-sensitive host cell line and determined termination sites in wild-type and nitA cells. We found that (i) normal phi X174 terminators were recognized in phi X174-infected nitA cells, (ii) the rho mutation relieved polar effects caused by nonsense mutations in the phage genome or by chloramphenicol treatment of the host cells, and (iii) polarity was not caused by premature termination of transcription at the site of the polar mutation. RNA synthesis continued beyond the site to the first rho-sensitive site.

Bacteriophage phi X 174↗
PubMed1992

Cytosine nucleoside inhibition of the ATPase of Escherichia coli termination factor rho: evidence for a base specific interaction between rho and RNA.

The function of rho factor in transcription termination depends on interactions with nascent RNA molecules that contain unpaired cytidylate residues. We show that cytidine, as a free nucleoside, inhibits the binding of rho to lambda cro mRNA and is a competitive inhibitor of rho-ATPase activity with lambda cro mRNA as cofactor. The relative ability of various cytidine analogs and other nucleosides to inhibit the rho-RNA interaction was used to probe features responsible for the base specificity of rho action. The results suggest that rho has a specificity pocket in its polynucleotide-binding site that apparently can make H-bond interactions with the side of the cytosine ring that normally faces away from the sugar ring and that may involve a relatively close fit along the edge of the ribose ring at the C2' carbon. The nature of the complex of rho with cytidine nucleotides was analyzed further by determining whether incubation with BrCMP caused inactivation of rho ATPase. Although BrCMP could form Michaelis inhibition complexes, it did not activate rho. Rho thus lacks a diagnostic property of enzymes that make specific covalent addition complexes with pyrimidines.

Adenosine Triphosphatases↗
PubMed1987

Transcription termination factor rho is an RNA-DNA helicase.

E. coli rho factor can unwind a short RNA-DNA duplex in vitro. The duplex is formed between a polylinker sequence at the 3' end of RNA derived from the rho-dependent terminator trp t' and the complementary sequence in a single-strand DNA molecule. Release of trp t' RNA from the duplex requires nucleoside triphosphate hydrolysis by rho's NTPase activity and is dependent on rho recognition of the RNA that is 5' to the RNA-DNA duplex region. The direction of helix unwinding appears to be 5' to 3' along the RNA molecule. These characteristics now account for how the RNA-binding and RNA-dependent NTP hydrolysis activities of rho may participate directly in transcription termination. Our results suggest that NTP hydrolysis is utilized to help unwind the RNA-DNA duplex at the 3' end of a nascent transcript, facilitating RNA release from the DNA template.

DNA Helicases↗
PubMed1990

Structural and functional properties of the segments of lambda cro mRNA that interact with transcription termination factor Rho.

Termination of transcription at tR1, the Rho-dependent terminator between genes cro and cII of bacteriophage lambda, is dependent upon the structure of segments near the 3' end of the nascent cro gene transcript and on contacts between Rho protein and a 3' proximal segment called rut. The characteristics of the structure of cro RNA in the region from residue 220 to residue 355 in free, isolated RNA and in the presence of Rho or NusA proteins were analyzed by measuring relative rates of reactivity of individual nucleotides with chemicals and enzymes of defined specificities. The results indicate that the rut segments are single-stranded and become blocked to the action of the various probes in the presence of Rho factor. They also show that this region contains two stem-loop structures; one involves the boxB sequence of nutR, the other precedes the tR1 subsite II end points. The results provide direct evidence for a primary binding contact between Rho protein and the rut segment of cro RNA and demonstrate that this binding contact remains stable when the cro RNA is serving as a cofactor for ATP hydrolysis, an observation that is consistent with a mechanism in which Rho maintains contact with the rut region while it makes additional interactions with RNA that are coupled to ATP hydrolysis.

Adenosine Triphosphate↗
PubMed2001

Transcription factor Rho does not require a free end to act as an RNA-DNA helicase on an RNA.

Escherichia coli Rho factor is a ring-shaped, homohexameric protein that terminates synthesis of RNA through interactions with the nascent RNA transcript. Because its mechanism of action may involve translocation of the RNA transcript through the hole in its ring structure, its action could depend on the availability of a free 5' terminus. To determine whether Rho's activity is 5'-end-dependent, its ability to bind to and function on a circular derivative of lambda cro mRNA was investigated. The circular derivative was made in vitro by action of RNA ligase on a derivative of lambda cro RNA containing an extra 10-nucleotide sequence near the 5'-end that was complementary to a sequence located near the 3'-end. Rho bound nearly as tightly to the circular derivative RNA as to the standard cro transcript. Rho was also able to readily dissociate a DNA oligonucleotide from its helical complex with the circular RNA in an ATP-dependent reaction. Thus, the action of Rho on a transcript does not depend on the availability of a free 5' terminus.

Adenosine Triphosphate↗
PubMed2000

RNA passes through the hole of the protein hexamer in the complex with the Escherichia coli Rho factor.

Escherichia coli transcription termination factor Rho is a ring-shaped hexameric protein that uses the energy derived from ATP hydrolysis to dissociate RNA transcripts from the ternary elongation complex. To test a current model for the interaction of Rho with RNA, three derivatives of Rho were made containing single cysteine residues and modified with a photo-activable cross-linker. The positions for the cysteines were: 1) in part of the primary RNA-binding site in the N terminus (Cys-82 Rho); 2) in a connecting polypeptide proposed to be on the outside of the hexamer (Cys-153 Rho); and 3) near the proposed secondary RNA-binding site in the ATP-binding domain (Cys-325 Rho). Results from the cross-linking of the modified Rho proteins to a series of lambda cro RNA derivatives showed that Cys-82 Rho formed cross-links with all transcripts containing the Rho utilization (rut) site, that Cys-325 Rho formed cross-links to transcripts that had the rut site and 10 or more residues 3' of the rut site, and that Cys-153 did not form cross-links with any of the transcripts. From a model of the quaternary structure of Rho, which is largely based on homology to the F(1)-ATPase, amino acid 82 is located near the top of the hexamer, and amino acid 325 is located on a solvent-accessible loop in the center of the hexamer. These data are consistent with binding of the rut region of RNA around the crown, with its 3'-segment passing through the center of the Rho hexamer.

Adenosine Triphosphate↗
PubMed1975

Termination of transcription in bacteriophage lambda. Heterogeneous, 3'-terminal oligo-adenylate additions and the effects of rho factor.

RNA transcripts were synthesized in vitro from a lambda DNA template with purified Escherichia coli RNA polymerase either in the presence or absence of the protein termination factor, rho. The products were initially characterized by electrophoresis on polyacrylamide slab gels, and two of the lower molecular weight discrete species (6 S and 4 S RNA) were further characterized by standard two-dimensional "fingerprint" analysis. Production of the 4 S RNA was strongly affected by the presence of rho, whereas production of the 6 S RNA species was relatively unaffected by rho. 3'-Terminal oligonucleotide fragments were then selectively isolated on columns of dihydroxyboryl-substituted cellulose from these transcripts. Sequence analysis of these oligonucleotide products indicated: (a) that all of the transcripts examined possess similar degrees of 3'-terminal sequence heterogeneity which consisted predominantly of the addition of 1 to 5 adenylate residues to the 3'-terminus of the transcript; and (b) that rho factor-enhanced termination results in a definite structural change in the nucleotide sequence with which an RNA molecule can terminate.

Adenine Nucleotides↗
PubMed1990

Specificity and efficiency of rho-factor helicase activity depends on magnesium concentration and energy coupling to NTP hydrolysis.

The RNA-DNA helicase activity of Escherichia coli transcription termination factor rho can be significantly enhanced at lower potassium chloride and magnesium acetate concentrations than previously used. Decreasing the potassium chloride concentration from 150 to 50 mM increases the rate of release at least 4-fold, while at lower magnesium concentrations less ATP is required for maximal duplex disruption. For all concentrations tested (between 0.1 and 5 mM), the optimal magnesium and ATP concentrations are interdependent; a roughly equimolar ratio gives the maximal rate of RNA release, although peak height and breadth vary. Surprisingly, rho behaves differently with an RNA-RNA duplex, which cannot be efficiently disrupted at magnesium concentrations below 1 mM. Above 2.0 mM, release does occur efficiently suggesting that Mg2+ promotes some structural transition in the RNA-RNA helix to a rho-susceptible conformation. In addition to Mg2+, helicase activity requires hydrolysis of nucleoside triphosphates, but for all four standard NTPs the rates of NTP hydrolysis do not correlate uniformly with the rates of RNA release. Based on the ratio of the rate of RNA release to the rate of NTP hydrolysis, rho utilizes ATP most efficiently. The 2-4-fold weaker coupling of hydrolysis to duplex disruption for the other three NTPs demonstrates that NTP utilization is not, on its own, sufficient for efficient helicase activity. The less efficient coupling with GTP, CTP, and UTP correlates with conformational differences in the protein complex as probed by mild trypsin digestion. The implications of our findings for substrate specificity and energy coupling in the helicase reaction are discussed.

Adenosine Triphosphatases↗
PubMed1990

A short intervening structure can block rho factor helicase action at a distance.

We have characterized the helicase activity of transcription termination factor rho on a variety of substrates. Helicase activity requires specific recognition of a single-stranded region of RNA upstream (5') of the nucleic acid duplex on which rho acts. Spacer sequences of at least 450 nucleotides can be inserted between the rho-binding signals and the duplex region with little effect on activity. RNA-DNA helices of up to 120 base pairs, but not as long as 210 base pairs, can be disrupted efficiently by rho. The stoichiometry of release of substrates with long spacer sequences, as with the standard substrate, approaches a value of one RNA released per rho hexamer; thus cooperative binding by rho does not account for action at a distance. Instead, these results are consistent with a model in which a single rho hexamer binds initially to terminator sequences and then either loops out or tracks along the intervening RNA to reach the duplex region. Results with complex substrates are inconsistent with looping and support the tracking model: under conditions that allow disruption of RNA-DNA, but not RNA-RNA helices (0.4 mM Mg2+), the presence of a short RNA-RNA helix acts as a block to the disruption of an RNA-DNA helix downstream. These findings are discussed in relation to the mechanism of the helicase activity as well as its role in rho-dependent transcription termination.

Adenosine Triphosphatases↗
PubMed1992

A vector for controlled, high-yield production of specifically mutated proteins in Escherichia coli: test of a putative cytidine-binding domain in Rho factor and its Thr16----Ala mutant.

A derivative of the plasmid vector, pET-3a [Rosenberg et al., Gene 56 (1987) 125-135], is described that contains the origin of replication from bacteriophage f1. This plasmid is well-suited for oligodeoxyribonucleotide mutagenesis and controlled production of mutant proteins from a single vector. Its utility is demonstrated by the preparation of a mutational alteration of Thr16----Ala (T16A) of the Escherichia coli transcription termination factor, Rho. The altered protein (T16A Rho) binds oligo(C)7 with the same affinity as wild-type (wt) Rho, thus indicating that Thr16 is not critical for binding cytidine residues in RNA, in spite of its being part of a sequence that is similar to a sequence in the CTP-binding domain of aspartate transcarbamoylase. However, T16A Rho was less efficient in terminating transcription than was wt Rho and had a lowered kcat for ATP hydrolysis with cro RNA as co-factor. Thus, the change affects the coupling of ATP hydrolysis by Rho to actions on RNA that cause termination.

Base Sequence↗
PubMed1984

Binding of rho factor to Escherichia coli RNA polymerase mediated by nusA protein.

The E. coli transcription termination factor rho binds specifically to purified nusA protein. Since nusA protein binds tightly to RNA polymerase, this provides a way of coupling rho to the elongating RNA polymerase complex through protein-protein interactions. These rho-nusA interactions may play a role in modulating rho action at certain terminators and could also be important in the action of antitermination factors such as lambda N protein.

Adenosine Triphosphatases↗