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D P Nierlich

Publications and source records attributed to D P Nierlich.

At least 19 recordsLinked to original sources
PubMed1991

Transcription and decay of the lac messenger: role of an intergenic terminator.

Prior work has indicated that the polycistronic lacZYA mRNA of Escherichia coli is cleaved during decay at approximately intergenic sites (L. W. Lim and D. Kennell, J. Mol. Biol. 135: 369-390, 1979). In this work, we characterized the products by using probes specific for the different cistrons. This analysis indicated that six lac mRNA species are present in the following order of decreasing abundance: lacZ, -A, -ZYA, -ZY, -YA, and -Y. Very little lacYA and lacY mRNAs were present, whereas in cells induced to steady state, there was 10 times more lacZ than lacZYA mRNA. The lacZ mRNA appeared as a discrete species extending to a site in the lacZ-Y intergenic space (ca. residue 3150). This site is just distal to a potential rho-independent termination sequence. We examined the function of this sequence to determine whether it contributes to the distribution of the mRNAs. Although the termination sequence was shown to function in vitro, when it was recloned into an expression vector, no termination was seen in vivo. Moreover, direct examination of the kinetics of lac messenger synthesis revealed that after initiation, most transcription continued to the end of the operon. We conclude that during normal growth, the operon is transcribed in its entirety and that the individual lac mRNAs are formed by cleavage. These results confirm earlier work implying that the lac operon is transcribed in its entirety but are in conflict with several recent reports suggesting that internal termination occurs. Our findings indicate that the natural polarity of the operon (lacZ is expressed sixfold more strongly than lacA) is based on posttranslational effects and not on polarity of transcription.

Base Sequence↗
PubMed1990

Use of the lac repressor in constructing sequential deletions and a new sequencing vector.

Large sequencing projects require an efficient strategy to generate a series of overlapping clones. This can be accomplished by protecting one end of a linear DNA molecule while sequential deletions are introduced into the other end by exonuclease digestion. We demonstrate that the lac repressor can protect the ends of linear nucleotide sequences from digestion by exonuclease if these ends contain the lac operator sequence. To exploit this, we have inserted the lac operator sequence between the primer-binding site and multiple cloning site of an M13 sequencing vector. Linearizing the replicative form and binding lac repressor protein protects the end next to the vector sequences. Sequential deletions are then introduced into the insert by digesting with exonuclease III or BAL 31. Because the rate and time of digestion are readily controlled, the region brought next to the sequencing primer site, after religation, can be selected in a timed series of reactions. This minimizes the screening needed to isolate an overlapping series of clones and facilitates sequencing of long regions.

Base Sequence↗
PubMed1989

Mapping the lacZ ribosome binding site by RNA footprinting.

The ribosome binding site of the Escherichia coli lacZ mRNA has been characterized by using an RNA footprinting technique. Purified E. coli 70S ribosomes and fMet-tRNA were incubated with mRNA, and the complex was treated with RNA-reactive reagents or RNases as probes. The protected sites on the mRNA were then mapped by extending a radioactive primer with reverse transcriptase. Dimethyl sulfate, diethyl pyrocarbonate, and 1,10-phenanthroline-copper ion oxidative complex were used as reagent probes; they detected interaction sites within the ribosome binding site. A region of approximately 35 nucleotides was protected by the ribosome, specifically across the Shine-Dalgarno region, around the fMet initiation codon, and at a region 7-12 nucleotides distal to the fMet codon. In addition, an enhanced reaction occurred between the fMet codon and the distal site. These results imply an internally selective interaction between the ribosome and the mRNA sequence. The enhanced reactivity of a site distal to the initiation site--flanked by the AUG codon and a site previously identified as conserved in a study of initiation sequences--may indicate a region where the mRNA is specifically exposed.

Alkylating Agents↗
PubMed1989

Scission of RNA by the chemical nuclease of 1,10-phenanthroline-copper ion: preference for single-stranded loops.

The scission of RNA by the chemical nuclease activity of 1,10-phenanthroline-copper (OP-Cu) has been studied using a lac mRNA fragment and tRNAphe as substrates. Since the chemical mechanism of scission involves oxidative attack on the ribose, scission is observed at all nucleotides including dihydrouridine and Y-bases. Specificity for single-stranded loop regions is apparent from the similarity of the reactivity of OP-Cu to the single-strand specific reagents dimethyl sulfate and diethyl pyrocarbonate using the fragment of lac mRNA as a substrate. Similar preference is observed in the reaction with tRNA although scission in the helical acceptor stem is also observed.

Base Sequence↗
PubMed1989

Distribution of 5'-triphosphate termini on the mRNA of Escherichia coli.

We have determined the distribution of 5'-nucleoside triphosphates on the RNA in Escherichia coli. These groups represent the initial nucleoside triphosphate incorporated when RNA polymerase initiates transcription. It was estimated that at least 15% of polysome-associated messengers had triphosphates. This was interpreted to mean that removal of the triphosphate or messenger leader is not necessary for the functioning of most mRNAs but that a substantial amount of messenger processing occurs in the polysome pool. We found that the ratio of GTP- to ATP-initiated messengers was about 2 to 1. Since prior work has indicated that G- and A-initiated RNAs decay at the same rate and since a compilation of messenger start sites shows an A preference, this value implies that there is a significant physiological selection of G-initiated transcripts. We also characterized the 5'-terminal groups on RNAs in other fractions. A small amount was found associated with 30S ribosomes, presumably in initiation complexes; such complexes have not previously been detected in situ. In addition, it was concluded that the 5' terminus of rRNA precursors is processed more rapidly than is implied by the current literature.

Base Composition↗
PubMed1987

Plotting genetic maps on a microcomputer.

Maps of genetic linkage and restriction enzyme cleavage sites can be quickly prepared on an IBM PC microcomputer with the commercially available program Lotus 1-2-3. Data can be entered on the keyboard or imported from other programs. The maps can be displayed on the screen or with a printer or plotter. These procedures should be useful in the research laboratory, in preparing figures for publication and in teaching.

Chromosome Mapping↗
PubMed1987

Expression of a tRNA gene in the context of the lacZ mRNA.

Fusions of the gene for tyrosine suppressor tRNA, tyrT(Sup3), and the lacZ gene of Escherichia coli were constructed such that the tRNA gene could be expressed from either its own promoter or that of the lac operon. These chimeras, carried on phage M13 vectors, were tested for the expression of the tRNA in E. coli. The tRNA gene was expressed on the order of 10-fold more weakly from the lac promoter than from its own promoter. To examine whether pausing or premature termination of transcription played a role in determining the relative strength, the fusions were tested in a variety of genetic backgrounds and under different physiological conditions that uncouple transcription and translation. The expression of the tRNA was not enhanced in backgrounds in which polarity was weakened or under the other conditions tested, although a dependence on nusB function was observed when the tRNA was transcribed from the lac promoter. These results indicate that pausing or premature termination of transcription did not play a role in the weak expression of the gene fusions. The results further suggest that the transcription of the tyrT gene does not normally require relief from polarity as imposed by any of the known transcriptional termination systems, in contrast to the antitermination system thought to be involved in the expression of the rRNAs.

Bacterial Proteins↗
PubMed1985

DNA sequence of the lactose operon: the lacA gene and the transcriptional termination region.

The lac operon of Escherichia coli spans approximately 5300 base pairs and includes the lacZ, lacY, and lacA genes in addition to the operator, promoter, and transcription termination regions. We report here the sequence of the lacA gene and the region distal to it, confirming the sequence of thiogalactoside transacetylase and completing the sequence of the lac operon. The lacA gene is characterized by use of rare codons, suggesting an origin from a plasmid, transposon, or virus gene. UUG is the translation initiation codon. A preliminary examination of 3' end of the lac messenger in the region distal to the lacA gene indicates several endpoints. A predominant one is located at the 3' end of a G + C-rich hairpin structure, which may be involved in termination of transcription or in post-transcriptional processing. An open reading frame of 702 base pairs is present on the complementary strand downstream from lacA.

Acetyltransferases↗
PubMed1979

Isolation of the transfer RNA genes of bacteriophage T4 and transfer RNA synthesis in vitro.

Non-glucosylated T4 DNA was restricted with the endonuclease EcoRI and the mixture of DNA fragments separated by gel electrophoresis and transcribed with purified Escherichia coli RNA polymerase. Three purified fragments were shown to act as templates for tRNA synthesis. A smaller fragment, shown to be hybridizable to 32P-labeled T4 tRNA was not transcribable. It was concluded that the promoter for T4 tRNA synthesis had been separated from the structural genes in the smaller fragment by EcoRI and that the distal portion of the tRNA gene cluster lacks internal promoters which display in vitro activity. Preparations of non-glucosylated T4 DNA were never fully restricted with EcoRI and when the larger purified fragments carrying the tRNA were restricted with excess enzyme only a slight cleavage to yield the smaller fragments was obtained. The property of the DNA-limiting complete restriction is not know.

Cell-Free System↗
PubMed1978

Yeast mutant, rna 1, affects the entry into polysomes of ribosomal RNA as well as messenger RNA.

The entry of newly labeled ribosomal subunits and mRNA into polysomes was examined in the yeast mutant rna1. The entry of both types of RNA into polysomes is inhibited rapidly at the restrictive temperature. Analysis of the labeling of the ATP pool and the kinetics of synthesis and processing of mRNA at the restrictive temperature leads to the conclusion that the primary defect in the mutant affects transport of both ribosomes and messenger across the nuclear membrane.

Adenosine Triphosphate↗
PubMed1976

Ribosomal subunit entry into polysomes in yeast.

The kinetics of entry of newly synthesized 40 S and 60 S ribosomal subunits into yeast polysomes is described. The entry times for 40 S and 60 S subunits were found to be 3 and 8 min, respectively. The kinetics of entry of 40 S subunits into large polysomes is found to be different from the kinetics of entry of 60 S subunits into large polysomes.

Adenine↗
PubMed1975

Regulation of RNA synthesis in Escherichia coli during a shift-up transition.

These experiments investigate two aspects of RNA synthesis in Escherichia coli ML30 during the transition from a relatively slow rate of growth to a more rapid one: (1) the number of growing RNA molecules per cell, and (2) the average time required for addition of a nucleotide onto a growing RNA chain. Cells were grown at 30 degrees C in a glucose-minimal salts medium and shifted-up by the addition of Casamino acids. Measurements were made of the rates of incorporation over short intervals (e.g. 5,8,12, and 16 s) of [3-H]guanine into the internal and 3'-terminal nucleotides of RNA. After correction for the specific activities of the intracellular GTP pools, and for the rate of [3-H]guanine accumulation at the 3'-terminus of non-growing RNA, the rates of chain elongation were calculated. It was found that cells growing at a rate of 0.9 generations/h contain approx. 4800 RNA molecules, growing at a rate of 28 nucleotides/s per chain. Cells growing exponentially at the postshift-up rate (1.2 generations/h) contain 7000 RNA molecules per unit equivalent cell mass, which are growing at a rate of 32 nucleotides/s per molecule. Three min after shift-up, cells contain the same number or slightly fewer (10%) growing RNA molecules than cells prior to shift-up, 4300, and these are being elongated at a rate of about 32 nucleotides per s. The results are consistent with the view that in the range of growth rates studied, the total rate of RNA synthesis is regulated through a limitation in the number of functioning RNA polymerase molecules, each working at a relatively constant, presumably maximal, average rate.

Cell Division↗
PubMed1975

Cleavage of Nonglucosylated Bacteriophage T4 deoxyribonucleic acid by Restriction Endonuclease Eco RI.

DNAs lacking the glucosyl modification (Glc-) and additionally lacking the 6-methylaminopurine (N6-methyladenine) modification (Glc-, MeAde-) were prepared from appropriate T4 mutants. These DNAs were cleaved by the purified restriction endonuclease Eco TI from Escherichia coli. Normally modified DNA (Glc+, MeAde+) was not attached. The Eco RII and the hemophilus enzymes Hin dII and Hin dIII do not attack Glc-, MeAde- T DNA, possibly due to the presence of 6-hydroxymethylcytosine. Eco RI produces approximately 40 specific fragments from Glc- DNA ranging in molecular weights from 0.3 to 10.5 X 10-6.

Adenine↗
PubMed1975

Initiation and transcription of a set of transfer ribonucleic acid genes in vitro.

Using RNA polymerase purified from Escherichia coli, DNA isolated from the bacteriophage T4, and a bacterial supernatant fraction containing the necessary processing enzymes, a set of transfer RNAs can be formed in vitro. To characterize the site or sites of initiation of this tRNA transcription, rifampicin-resistant complexes of RNA polymerase, DNA, and either ATP (UTP and CTP) or GTP (UTP and CTP) were formed, and tRNA was transcribed from these stabilized sites. It is concluded that transcription of the entire set is initiated by ATP. To study the transcription of the tRNAs, the time sequence of the appearance of individual species was determined during synchronous transcription of a preformed RNA polymerase-DNA complex. The appearance of three RNA species is found to be consistent with the sequential transcription of a large polycistronic cluster; the order and distances, inferred from the times of transcription, are as required by the existing gene map. It is concluded that the initiation of tRNA transcription can occur, without accessory factors, with the insertion of ATP at a single or a few closely spaced sites, and that the tRNAs encoded by the bacteriophage T4 are present in a single operon.

Chromosome Mapping↗