http://wolfson.huji.ac.il/expression/local/rec-exp-99.pdf

Recombinant protein expression in Escherichia coli.

https://mmbr.asm.org/content/mmbr/47/1/1.full.pdf

Comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles.

Formation of mRNA 3′ ends in eukaryotes requires the interaction of transacting factors with cis-acting signal elements on the RNA precursor by two distinct mechanisms, one for the cleavage of most replication-dependent histone transcripts and the other for cleavage and polyadenylation of the majority of eukaryotic mRNAs. Most of the basic factors have now been identified, as well as some of the key protein-protein and RNA-protein interactions. This processing can be regulated by changing the levels or activity of basic factors or by using activators and repressors, many of which are components of the splicing machinery. These regulatory mechanisms act during differentiation, progression through the cell cycle, or viral infections. Recent findings suggest that the association of cleavage/polyadenylation factors with the transcriptional complex via the carboxyl-terminal domain of the RNA polymerase II (Pol II) large subunit is the means by which the cell restricts polyadenylation to Pol II transcripts. The processing of 3′ ends is also important for transcription termination downstream of cleavage sites and for assembly of an export-competent mRNA. The progress of the last few years points to a remarkable coordination and cooperativity in the steps leading to the appearance of translatable mRNA in the cytoplasm.

Formation of mRNA 3′ Ends in Eukaryotes: Mechanism, Regulation, and Interrelationships with Other Steps in mRNA Synthesis

http://teachline.ls.huji.ac.il/72682/Publications/Sorensen2005.pdf

Advanced genetic strategies for recombinant protein expression in Escherichia coli.

Sporulation of Bacillus subtilis

Many bacterial mRNAs, like those of eukaryotes, carry a polyadenylate sequence at their 3' termini, but neither the function of the bacterial poly(A) moieties nor their biosynthesis have been elucidated. To develop a genetic tool to approach the problem of bacterial poly(A) RNA, we have sought to identify the genes responsible for mRNA polyadenylylation. A poly(A) polymerase was purified to homogeneity from extracts of Escherichia coli and subjected to N-terminal sequence analysis. The 25-residue amino acid sequence obtained was used to design primers for the amplification of the corresponding coding region by the PCR from an E. coli DNA template. A 74-base-pair DNA segment was obtained that matched a region in the pcnB locus of E. coli, a gene that had originally been identified as controlling plasmid copy number [J. Lopilato, S. Bortner & J. Beckwith (1986) Mol. Gen. Genet. 205, 285-290] and was subsequently cloned and sequenced [J. Liu & J. S. Parkinson (1989) J. Bacteriol. 171, 1254-1261]. Direct evidence that the pcnB locus encodes poly(A) polymerase was provided by the observation that a bacterial strain transformed with an inducible expression vector carrying pcnB as a translational fusion produced 100-fold elevated levels of poly(A) polymerase upon induction. No increased poly(A) polymerase activity was observed in cells transformed with expression vectors carrying truncated forms of the pcnB gene. The identification of a gene encoding bacterial poly(A) polymerase opens the way for the study of the biosynthesis and function of bacterial polyadenylylated mRNA.

https://www.pnas.org/content/pnas/89/21/10380.full.pdf

Identification of the gene for an Escherichia coli poly(A) polymerase.

Tyrothricin specifically inhibits RNA synthesis in growing cultures of Bacillus brevis as well as purified RNA polymerase. This suggests that the peptide antibiotic may function in the regulation of gene transcription during the transition from vegetative growth to sporulation.

Function of peptide antibiotics in sporulation.

The Methylation of Lysine Residues in Protein

A new one-step procedure for the isolation of bacterial RNA, involving lysis by proteinase K in the presence of sodium dodecyl sulfate, is described. Pulse-labeled RNA isolated by this procedure for Bacillus brevis, Bacillus subtilis, and Escherichia coli B has been found to contain a substantial fraction (15-40%) of polyadenylated RNA as determined by adsorption to oligo(dT)-cellulose. This contrasts with RNA isolated by procedures involving phenol extraction, a process which appears to lead to the selective loss of polyadenylated RNA. The presence of polyadenylated RNA in E. coli was confirmed by an independent method which involved hybridization with [3H]polyuridylic acid. Using the proteinase K method for RNA isolation, it was possible to demonstrate the in vitro synthesis of polyadenylated RNA by toluene-treated cells of B. brevis, B. subtilis, and E. coli.

/pdf/detection-of-high-levels-of-polyadenylate-containing-rna-in-16004p9xa5.pdf

Detection of high levels of polyadenylate-containing RNA in bacteria by the use of a single-step RNA isolation procedure

We had earlier identified the pcnB locus as the gene for the major Escherichia coli poly(A) polymerase (PAP I). In this report, we describe the disruption and identification of a candidate gene for a second poly(A) polymerase (PAP II) by an experimental strategy which was based on the assumption that the viability of E. coli depends on the presence of either PAP I or PAP II. The coding region thus identified is the open reading frame f310, located at about 87 min on the E. coli chromosome. The following lines of evidence support f310 as the gene for PAP II: (i) the deduced peptide encoded by f310 has a molecular weight of 36,300, similar to the molecular weight of 35,000 estimated by gel filtration of PAP II; (ii) the deduced f310 product is a relatively hydrophobic polypeptide with a pI of 9.4, consistent with the properties of partially purified PAP II; (iii) overexpression of f310 leads to the formation of inclusion bodies whose solubilization and renaturation yields poly(A) polymerase activity that corresponds to a 35-kDa protein as shown by enzyme blotting; and (iv) expression of a f310 fusion construct with hexahistidine at the N-terminus of the coding region allowed purification of a poly(A) polymerase fraction whose major component is a 36-kDa protein. E. coli PAP II has no significant sequence homology either to PAP I or to the viral and eukaryotic poly(A) polymerases, suggesting that the bacterial poly(A) polymerases have evolved independently. An interesting feature of the PAP II sequence is the presence of sets of two paired cysteine and histidine residues that resemble the RNA binding motifs seen in some other proteins.

https://www.jstor.org/stable/pdfplus/40477.pdf

Nilima Sarkar

Papers

Identification of the gene for an Escherichia coli poly(A) polymerase.

Function of peptide antibiotics in sporulation.

The Methylation of Lysine Residues in Protein

Detection of high levels of polyadenylate-containing RNA in bacteria by the use of a single-step RNA isolation procedure

Identification of the coding region for a second poly(A) polymerase in Escherichia coli.