Nilima Sarkar

Studies on the attachment and release of 5 s ribosomal RNA from the large ribosomal subunit

Abstract The 80 s ribosomes of Blastocladiella emersonii or the 50 s ribosomal subunits of Escherichia coli RNase − 10 exchange their 5 s ribosomal RNA with externally added 5 s rRNA at 0.1 m m -Mg 2+ but at 10m m exchange is greatly inhibited. Very high external concentrations of 5 s rRNA are required to demonstrate exchange. When Mg 2+ is partially removed from the large ribosomal subunit of E. coli with EDTA, 5 s rRNA is released when the particle is unfolded to a 33 to 36 s particle. The 33 to 36 s particles are capable of binding 5 s rRNA at 0.1 m m -Mg 2+ but the addition of protein extracted from 50 s particles, stimulates the binding of 5 s at low ratios of 5 s per particle. Particles from which 5 s has been partially or completely removed are inactive in in vitro polyphenylalanine synthesis directed by polyuridylic acid. Reassociation with 5 s did not restore activity. Unfolded particles without 5 s can be converted to a more compact 47 s particle by heating to 50 °C for 10 minutes in 10 m m -Mg 2+ buffer.

The binding of 5 s ribosomal ribonucleic acid to ribosomal subunits

Abstract The 75 s ribosomes of the aquatic fungus, Blastocladiella emersonii dissociate at 10 −4 m -Mg 2+ into 63 s and 45 s subunits. Only the 63 s subunit contains 5 s rRNA, about one molecule per subunit being present. When these subunits are treated with EDTA, the 63 s particle is unfolded to a 47 s particle without extensive release of protein but with the release of 5 s rRNA. Using 32 P-labeled 5 s rRNA, we have demonstrated that 5 s rRNA will exchange with the 5 s rRNA on the 63 s subunit. Binding studies demonstrate that 2 molecules of 5 s rRNA will bind to ribosomal subunits or to the isolated 47 s large subunit. Binding to one of the two sites is displaced by transfer RNA.

Bacteriophage T7 mRNA is polyadenylated

To determine whether the RNA of bacterial viruses is polyadenylated like bacterial mRNAs, pulse‐labelled as well as the steady‐state population of bacteriophage T7‐specific transcripts were examined for the presence of poly(A) tracts by binding to oligo(dT) cellulose followed by hybridization with specific gene probes. Representatives of all classes of bacteriophage‐specific mRNA — early, middle and late — were found to be polyadenylated. This conclusion was confirmed by screening the products of oligo(dT)‐dependent cDNA synthesis. A cDNA library was prepared from RNA synthesized after bacteriophage T7 infection and the sequence of bacteriophage‐specific clones was determined to define the sites of polyadenylation. About half of the clones were polyadenylated near the end of a protein‐coding region, one of them at the site of post‐transcriptional processing by RNase III. Other clones were polyadenylated within protein‐coding regions. These observations suggest that polyadenylation occurs after the nucleolytic processing of primary transcripts and in some cases also after mRNA degradation has already begun.

Construction of a cDNA library from polyadenylated RNA of Bacillus subtilis and the determination of some 3′-terminal sequences

We had found previously that polyadenylated RNA constitutes a surprisingly large fraction of mRNA in both Escherichia coli and Bacillus subtilis [Gopalakrishna et al., Nucl. Acids Res. 9 (1981) 3545-3554; Biochem. 21 (1982) 2724-2729]. We have also shown [Gopalakrishna and Sarkar, J. Biol. Chem. 257 (1982) 2747-2750] that polyadenylated RNA from B. subtilis can serve as a template for the synthesis of complementary DNA by reverse transcriptase using oligo(dT) as primer. In this work, we show that the cDNA thus synthesized contains sequences representative of poly(A)+RNA and can serve as template for double-stranded (ds) cDNA synthesis. The ds cDNA could be inserted into the PstI site of pBR322 and cloned in E. coli DH1. The cDNA inserts from a few cloned recombinant pBR322 plasmids were transferred to M13mp18 bacteriophage for sequence determination. Six cDNA species had terminal oligo(dT) sequences, indicating that they represented the complement of poly(A)+RNA. This constitutes independent and direct evidence for the existence of bacterial polyadenylated mRNA and opens the way for studying the nucleotide sequences that control polyadenylation.

Identification of two poly(A) polymerases in Bacillus subtilis.

Two poly(A) polymerase activities were identified in extracts of a strain of Bacillus subtilis in which the gene for polynucleotide phosphorylase was disrupted. Gel filtration studies showed a large difference in the molecular size of the two poly(A) polymerases. On the other hand, the two enzymes resembled the two major poly(A) polymerases of Escherichia coli both with respect to size and in many of their catalytic properties. The observation that both B. subtilis and E. coli have two poly(A) polymerases with many common properties suggest interesting parallels in the processing of the 3′‐ends of mRNA in gram‐positive and gram‐negative bacteria.

Selective resistance of bacterial polyadenylate-containing RNA to hydrolysis by guanosine 3′-5′-monophosphate-sensitive nuclease of Bacillus brevis

Abstract Earlier experiments had shown that the degradation of newly synthesized RNA in permeable cells of Bacillus brevis is mediated primarily by a guanosine 3′,5′-monophosphate-sensitive 3′-exonuclease [ N. Sarkar and H. Paulus (1975) J. Biol. Chem. 250, 684–690]. More recently, we found that a substantial fraction of pulse-labeled RNA in B. brevis is polyadenylylated [ N. Sarkar, D. Langley, and H. Paulus (1978) Biochemistry 17, 3468–3474], and it was thus of interest to examine the effect of polyadenylylation on the susceptibility of RNA to degradation by the 3′-exonuclease. Purified 3′-exonuclease from B. brevis hydrolyzed the unadenylylated fraction of pulse-labeled RNA from B. brevis much more rapidly than poly(A)-containing RNA. Similar results were obtained with the pulse-labeled unadenylylated and polyadenylylated RNA fractions from Bacillus subtilis . Control experiments showed that the differential hydrolysis of the labeled RNA preparations by 3′-exonuclease was not due to the presence of inhibitors or activators. These results suggest that the stability of mRNA in Bacillus species may be regulated by polyadenylylation.

Involvement of RNA polymerase in the synthesis of DNA by growing and toluene-treated cells of bacillusbrevis*

Summary The incorporation of [ 3 H] thymidine into DNA by growing cultures of bacillus brevis as well as other Bacilli was strongly inhibited by rifampicin and streptolydigin, suggesting a role for RNA polymerase in DNA replication. On the other hand, DNA synthesis in toluene-treated cells of B . brevis , which was dependent on the presence of ATP or other ribonucleoside triphosphates, was unaffected by streptolydigin or rifampicin. However, upon storage of toluene-treated cells at −20°, sensitivity to inhibition by streptolydigin developed progressively (up to 76% inhibition) over a period of 4 weeks, while the overall rate of DNA synthesis remained constant.

Control of Transcriptionin Bacillus brevis by Small Molecules

Bacterial sporulation consists of a progression of biochemical changes whose orderly sequence seems to be effected primarily by the regulation of specific gene transcription (1). In order to understand the control mechanisms involved in this process, we have concentrated our attention on the ways by which RNA synthesis in Bacillus brevis may be modulated by low molecular weight substances.

Deoxyribonucleic acid replication in fetal cells

OBJECTIVES Our purpose was to develop a sensitive method for assessing the replication time of specific human genes in cultured fetal cells and for detecting potential replication defects. STUDY DESIGN Synchronous progression of diploid human fetal lung cells through S phase was achieved by releasing from serum restriction with minimum essential medium alpha modification plus 10% fetal bovine serum, followed by hydroxyurea blockage at the G1/S boundary. Deoxyribonucleic acid replication was studied in permeabilized cells using mercurated nucleotides to label nascent deoxyribonucleic acid. RESULTS A high degree of synchrony in traversal of S phase was indicated by flow cytometry and a well-defined 7-hour period of deoxyribonucleic acid synthesis. The replication of the topoisomerase II gene occurred in a narrow time span 3 hours after entry into S phase. CONCLUSIONS Fetal cells have been highly synchronized at the beginning of S phase, and the replication time of a specific gene can be defined within a narrow time window.

HALF-LIFE OF ESCHERICHIA COLI POLYADENYLATED LIPOPROTEIN MRNA

In the light of recent evidence that the half‐life of bacterial mRNA may be modulated by polyadenylation at the 3′ end, we determined the half‐life of polyadenylated lpp mRNA, which is an abundant and comparatively stable message encoding a major lipoprotein of the outer membrane. Messenger RNAs were pulse‐labeled with [3H] adenosine and poly(A) RNA was isolated at various times after pulse‐labeling by affinity chromatography on oligo(dT) cellulose. The amount of lpp mRNA remaining was quantitated by hybridization with the antisense strand of the lpp gene cloned in M 13mp18. The observed half‐life for polyadenylated lpp mRNA was 12 min. This represents the first half‐life measurement for a specific polyadenylated mRNA in E. coli, and is slightly longer than the half‐life of total lpp RNA reported earlier. It coincides with the functional half‐life for lpp mRNA determined by Inouye and coworkers by measuring the rate of lipoprotein synthesis at various times after rifampicin addition. This suggests that polyadenylated lpp RNA is the predominant and translationally active form of lpp mRNA within the cell.