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T Platt

Publications and source records attributed to T Platt.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1986

The rho-115 mutation in transcription termination factor rho affects its primary polynucleotide binding site.

We have investigated the effect of the rho-115 mutation on the catalytic properties of the Escherichia coli termination protein, rho. Comparison of the primary and secondary polynucleotide binding sites activities reveals dramatic differences between the mutant and wild-type molecules. Wild-type rho must bind single-stranded polynucleotides to activate its nucleotide triphosphatase (NTPase) activity, and either poly(C), or poly(dC) plus oligo(C), will suffice. In contrast, attempted activation of the rho-115 NTPase with poly(C) in the presence of poly(dC) showed the latter to be a potent inhibitor. Inclusion of small oligonucleotides such as oligo(C) in the activation assay does not inhibit the poly(C)-induced NTPase reaction of either wild-type rho or rho-115. This would indicate, in the two polynucleotide binding site model for rho proposed by Richardson (Richardson, J.P. (1982) J. Biol. Chem. 251, 5760-5766), that the mutation in rho-115 affects the primary polynucleotide binding site. Transcription termination in vitro at the rho-dependent site trp t' showed dramatically reduced termination with rho-115 protein compared to wild-type rho. In the presence of rho-115, the transcript is longer and termination occurs over a narrower range of nucleotides than with wild-type rho. This suggests that the primary polynucleotide binding site is important not only for efficient termination of transcription but may also be involved in determining the terminal end point of the transcript itself.

Binding Sites↗
PubMed1985

Maturation of Escherichia coli tryptophan operon mRNA: evidence for 3' exonucleolytic processing after rho-dependent termination.

The mature 3' end of Escherichia coli tryptophan operon mRNA in vivo coincides with a site (trp t) having features commonly associated with rho-independent terminators in bacteria. Efficient generation of this 3' end in vivo is nevertheless affected by a distal rho-dependent site (trP-t'), though these two sites behave independently in vitro. We have cloned these sites upstream of the galactokinase gene (galK), and galactokinase levels in vivo indicate that, as terminators per se, their efficiencies (37% for trp t, and 79% for trp t') do not differ significantly from those observed in vitro. However, when the trp t hairpin is placed between galK and a downstream copy of trp t', galactokinase levels are enhanced 2- to 3-fold. This suggests the involvement of a post-transcriptional event, such as RNA processing, in determining the level of gene activity. Indeed, in the presence of the 3' exonuclease RNase II, mRNA terminated by rho factor in vitro at the trp t' site is processed back to the trp t site. The remote trp t' region appears to be the major termination site for trp mRNA, and the trp t hairpin serves a dual function-as a minor terminator, and as a protective barrier to 3' exonucleolytic degradation. We infer that the tandem terminators, rho factor, and RNA processing are all required to generate the mature 3' end of this bacterial mRNA.

Base Sequence↗
PubMed1985

Maximizing gene expression from plasmid vectors containing the lambda PL promoter: strategies for overproducing transcription termination factor rho.

We have constructed two plasmids in which transcription of the rho gene from Escherichia coli K-12 is under the control of the lambda phage PL promoter. In p31-356, the normal rho promoter is deleted, but the remainder of the rho leader region, including the ribosome binding site, is present. In p39-AS, the rho leader is completely absent, and the lambda cII ribosome binding site replaces that of rho. Under noninducing conditions, expression of rho protein from these plasmids is repressed by the lambda cI protein in hosts carrying lambda cryptic prophage. Induction using mitomycin C or nalidixic acid in a cryptic lysogen carrying the cI+ repressor resulted in the overproduction of rho protein to levels of 3%-5% of the total cellular protein with p31-356, and to levels of approximately equal to 40% with p39-AS. The overproduced protein is functionally indistinguishable from the rho protein isolated from the K-12 strain W3110, and it can be obtained from cells harboring p39-AS in yields of up to 25 mg of rho per g of cells. In contrast to chemical induction, heat induction in four cryptic lambda lysogens carrying the thermolabile cI857 repressor failed to yield the same high levels of rho protein (with either plasmid). Our results show that chemical induction of PL-containing plasmid expression vectors can serve as a convenient and useful alternative to the commonly used method of heat induction.

Bacteriophage lambda↗
PubMed1985

Escherichia coli transcription termination factor rho has a two-domain structure in its activated form.

Limited tryptic digestion of Escherichia coli transcription termination factor rho [an RNA-dependent nucleoside triphosphatase (NTPase)] yields predominantly two fragments (f1 and f2) when the protein is bound to both poly(C) and ATP. The apparent molecular masses of the two fragments are 31 kDa for f1 and 15 kDa for f2, adding up to the molecular mass of the intact rho polypeptide chain (46 kDa). Sequence analysis of the amino termini demonstrates that f1 is derived from the amino-terminal portion of rho and that the trypsin cleavage that defines f2 occurs at lysine-283. These results suggest that, in the liganded (activated) form, the native rho protein monomer is organized into two distinct structural domains that are separable by a single proteolytic cleavage. The f1 fragment, purified from NaDodSO4/polyacrylamide gels and renatured, binds poly(C) but the f2 fragment does not; neither regains any ATPase activity. ATP- and polynucleotide-dependent changes in the rate of proteolysis and in the character of the fragments produced suggest that rho undergoes a series of conformational transitions as a consequence of RNA binding, NTP binding and NTP hydrolysis. The rate of loss of rho ATPase activity and of intact rho monomers is slower in the presence of adenosine 5'-[gamma-thio]triphosphate than in the presence of either ATP or ADP, indicating that the hydrolysis of ATP may result in different conformational effects than does the binding of this ligand. These findings are discussed within the context of recent models of rho-dependent transcription termination.

Adenosine Triphosphate↗
PubMed1984

Targeted mutagenesis in vitro: lac repressor mutations generated using AMV reverse transcriptase and dBrUTP.

We have cloned the gene for the lac operon repressor (lacI) of Escherichia coli into the M13 related phage f1. Mutagenesis of the lacI gene was performed in vitro by filling dsDNA molecules gapped over the lacI gene with Avian Myeloblastosis Virus (AMV) reverse transcriptase. LacI mutants are found at a frequency of 1 in 10(4) using a genetic screen in vivo. For two-thirds of the 60 mutants, lesions were identified within the first 400 bases of lacI, by dideoxy sequencing. An unexpectedly wide range of different lesions were observed, including transitions, transversions, and deletions (of which the most common were the removal of single base pairs). The replacement of dTTP by dBrUTP in the filling reaction resulted in a doubling of deletions in the sample population as well as the anticipated T to C and C to T transitions. Although the lacI gene has been extensively studied in vivo, the power of this technique for mutagenesis in vitro is demonstrated by the generation of three previously undescribed lacI mutations.

Amino Acid Sequence↗
PubMed1984

Rho-dependent termination and concomitant NTPase activity requires a specific, intact RNA region.

We have investigated the specific DNA and RNA requirements for rho-dependent transcription termination in vitro. As a model, we have used templates containing the rho-dependent terminator of the Escherichia coli trp operon, trp t'. Templates containing the trp t' region direct specific rho-dependent termination in vitro, with concomitant stimulation of the rho NTPase activity, and deletion of the trp t' region results in templates that do not induce rho-dependent termination or rho NTPase activity. Addition of ribonuclease T1 to transcription reactions specifically eliminated transcription termination and rho NTPase activity. These results demonstrate the requirement for a specific RNA component within the trp t' transcript necessary for NTPase activation and rho-dependent transcription termination. Active transcription is not a prerequisite for rho NTPase activation; trp t' RNA (rho-terminated transcripts) and read-through transcripts, which contain the trp t' region, activated the rho NTPase when rho was added after inhibition of transcription. As is true for synthetic polynucleotides known to activate the rho NTPase, the trp t' region has few G residues. This reduces the potential for the formation of stable secondary structures in the RNA transcript, and may be one determinant of sites specifying rho-dependent termination of transcription. The implications of this are discussed in the light of the lack of significant sequence homologies between rho-dependent transcription termination sites.

Adenosine Triphosphatases↗
PubMed1984

Toxicity of 2-deoxygalactose to Saccharomyces cerevisiae cells constitutively synthesizing galactose-metabolizing enzymes.

Analysis of 400 independent spontaneous mutations conferring 2-deoxygalactose resistance upon cells constitutive for the galactose pathway suggests that toxicity is due to 2-deoxygalactose-1-phosphate. Selection for and against growth on galactose in the same strain is now possible; application to systems with transcriptional or translational gene fusions to galactokinase are discussed.

Carbohydrate Epimerases↗
PubMed1983

Nucleotide sequence of the trpD and trpC genes of Salmonella typhimurium.

We have completed the nucleotide sequence determination of trpD and trpC, the second and third genes of the trp operon of Salmonella typhimurium. These genes encode two bifunctional proteins thought to have arisen by gene fusions: the trpD polypeptide contains the glutamine amido transferase and the phosphoribosyl anthranilate transferase activities, and the trpC protein possesses the N-(5'-phosphoribosyl)-anthranilic acid isomerase and the indole-3-glycerol phosphate synthetase activities. The trpD gene consists of 1593 nucleotides encoding 531 amino acids, and possesses an internal promoter (p2) located within a region from about 1400 to 1441 of the nucleotide sequence. The trpC gene contains 1356 nucleotides encoding 452 amino acids. In this paper we compare the trpD and trpC genes of S. typhimurium to those of Escherichia coli with respect to codon usage, nucleotide and amino acid conservation, p2 promoter characteristics and intercistronic regions. The sequence of the two genes we present here completes the sequence determination of the trp operon of S. typhimurium and should prove useful in comparisons with the E. coli trp operon and in future studies of operon structure in S. typhimurium.

Amino Acid Sequence↗
PubMed1983

Initiation in vivo at the internal trp p2 promoter of Escherichia coli.

We have identified an RNA transcript initiated in vivo at the internal promoter of the Escherichia coli trp operon. The 5' end of this message overlaps the distal portion of the trpD structural gene, and the startpoint of transcription is the same as that previously determined in vitro. The relative abundance of the primary and secondary promoter transcripts in cells grown under varying conditions confirms previous genetic data suggesting that the p2 promoter is expressed at a low level, but constitutively. Comparison of p2 with a number of other recently identified internal promoters suggests that the primary function of these elements may be to provide a differentially regulated source of transcription for a subset of genes within the operon.

Base Sequence↗
PubMed1983

The nucleotide sequence of the rho gene of E. coli K-12.

We have determined the nucleotide sequence of the rho gene which encodes the E. coli K-12 transcription termination factor. The structural gene was located on a cloned 3.6 kilobase BglII-HindIII restriction fragment by the introduction of the insertion element gamma delta and analysis of the recombinant plasmids by restriction analysis and in maxicells. The coding region consists of 1260 nucleotides directing the synthesis of a polypeptide 419 amino acids in length with a calculated molecular weight of 46,094. The deduced amino acid composition, amino-terminal protein sequence and calculated molecular weight are consistent with the data from the analysis of purified rho protein (16). We have shown that the rho genes from E. coli K-12, B and C strains are located on PvuII-HindIII fragments of the same size by hybridization to the rho (K-12) coding sequences.

Amino Acid Sequence↗
PubMed1983

A kinetic mechanism for the poly(C)-dependent ATPase of the Escherichia coli transcription termination protein, rho.

A study of the initial velocity kinetics of the Escherichia coli transcription termination protein rho, with respect to its poly(C)-dependent ATPase, indicates that this reaction occurs by an ordered sequential mechanism. Product inhibition and substrate analogue studies suggest that ATP binding must precede the binding of poly(C) and that the order of release of the products is ADP followed by Pi, then poly(C). A possible mechanism for relating the ATPase to the termination reaction of rho is discussed in relation to the model for rho proposed by Richardson (5).

Adenosine Diphosphate↗
PubMed1982

Regulation of transcription from tandem and convergent promoters.

We have examined transcription on templates containing the trp and lac UV5 promoters arranged in tandem or opposing orientations. These studies have revealed that the strengths of the two promoters are comparable, though the lac UV5 promoter is much more sensitive to the level of initiating purine present. Kinetic experiments have shown that a polymerase molecule poised at the lac promoter, or a lac repressor molecule bound to the lac operator, can temporarily block a polymerase molecule initiated from the trp promoter, though transcription eventually continues through. In the convergent construct, transcription from the lac promoter is hindered only when initiation is suboptimal due to low purine concentrations.

Base Sequence↗