Interaction of Escherichia coli RNA Polymerase with the Eukaryotic TATA-Binding Protein

Interaction with eukaryotic TATA-binding protein (TBP) was analyzed for natural Escherichia coli RNA polymerase or the recombinant holoenzyme, minimal enzyme, or its subunit. Upon preincubation of full-sized RNA polymerase with TBP and further incubation with a constant amount of a ³²P-labeled phosph-amide derivative of a TATA-containing oligodeoxyribonucleotide, the yield of the holoenzyme–oligonucleotide covalent complex decreased with increasing TBP concentration. This was considered as indirect evidence for complexing of RNA polymerase with TBP. In gel retardation assays, the holoenzyme, but neither the minimal enzyme nor the subunit, interacted with TPB, since the labeled probe formed complexes with both proteins in the reaction mixture combining TBP with the minimal enzyme or the subunit. It was assumed that E. coli RNA polymerase is functionally similar to eukaryotic RNA polymerase II, and that the complete ensemble of all subunits is essential for the specific function of the holoenzyme.     

Characterization of Soluble Hepatitis C Virus RNA-Dependent RNA Polymerase Expressed in Escherichia coli

Production of soluble full-length nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) has been shown to be problematic and requires the addition of salts, glycerol, and detergents. In an effort to improve the solubility of NS5B, the hydrophobic C terminus containing 21 amino acids was removed, yielding a truncated NS5B (NS5BΔCT) which is highly soluble and monodispersed in the absence of detergents. Fine deletional analysis of this region revealed that a four-leucine motif (LLLL) in the hydrophobic domain is responsible for the solubility profile of the full-length NS5B. Enzymatic characterization revealed that the RNA-dependent RNA polymerase (RdRp) activity of this truncated NS5B was comparable to those reported previously by others. For optimal enzyme activity, divalent manganese ions (Mn²⁺) are preferred rather than magnesium ions (Mg²⁺), whereas zinc ions (Zn²⁺) inhibit the RdRp activity. Gliotoxin, a known poliovirus 3D RdRp inhibitor, inhibited HCV NS5B RdRp in a dose-dependent manner. Kinetic analysis revealed that HCV NS5B has a rather low processivity compared to those of other known polymerases.     

Expression of Enzymatically Active Rabbit Hemorrhagic Disease Virus RNA-Dependent RNA Polymerase in Escherichia coli

The rabbit hemorrhagic disease virus (RHDV) (isolate AST/89) RNA-dependent RNA-polymerase (3D^pol) coding region was expressed in Escherichia coli by using a glutathione S-transferase-based vector, which allowed milligram purification of a homogeneous enzyme with an expected molecular mass of about 58 kDa. The recombinant polypeptide exhibited rifampin- and actinomycin D-resistant, poly(A)-dependent poly(U) polymerase. The enzyme also showed RNA polymerase activity in in vitro reactions with synthetic RHDV subgenomic RNA in the presence or absence of an oligo(U) primer. Template-size products were synthesized in the oligo(U)-primed reactions, whereas in the absence of added primer, RNA products up to twice the length of the template were made. The double-length RNA products were double stranded and hybridized to both positive- and negative-sense probes.     

Control of ribosomal RNA synthesis in Escherichia coli. III. Cytoplasmic factors for ribosomal RNA synthesis.

The ribosomal RNA synthesis in a cell-free system containing the nucleoids and the cytoplasmic fraction prepared from Escherichia coli cells has been investigated. The addition of the "4S" fraction from the cytoplasm to the isolated nucleoids induces RNA synthesis by a new chain initiation. In this system a preferential initiation or rRNA chains occurs. The experimental results suggest that the 4S fraction contains at least two activities, one for releasing RNA-polymerases from the nucleoids, and another for the frequent initiation of rRNA chains. No restriction of the rRNA synthesis has been observed in the nucleoids and the 4S fraction from the amino acid-starved rel+ cells. The rRNA synthesized in the above system is detected at about 23S and 16S rRNA regions.     

Cytoplasmic protein mobility in osmotically stressed Escherichia coli

Facile diffusion of globular proteins within a cytoplasm that is dense with biopolymers is essential to normal cellular biochemical activity and growth. Remarkably, Escherichia coli grows in minimal medium over a wide range of external osmolalities (0.03 to 1.8 osmol). The mean cytoplasmic biopolymer volume fraction ((phi)) for such adapted cells ranges from 0.16 at 0.10 osmol to 0.36 at 1.45 osmol. For cells grown at 0.28 osmol, a similar phi range is obtained by plasmolysis (sudden osmotic upshift) using NaCl or sucrose as the external osmolyte, after which the only available cellular response is passive loss of cytoplasmic water. Here we measure the effective axial diffusion coefficient of green fluorescent protein (D(GFP)) in the cytoplasm of E. coli cells as a function of (phi) for both plasmolyzed and adapted cells. For plasmolyzed cells, the median D(GFP) (D(GFP)(m)) decreases by a factor of 70 as (phi) increases from 0.16 to 0.33. In sharp contrast, for adapted cells, D(GFP)(m) decreases only by a factor of 2.1 as (phi) increases from 0.16 to 0.36. Clearly, GFP diffusion is not determined by (phi) alone. By comparison with quantitative models, we show that the data cannot be explained by crowding theory. We suggest possible underlying causes of this surprising effect and further experiments that will help choose among competing hypotheses. Recovery of the ability of proteins to diffuse in the cytoplasm after plasmolysis may well be a key determinant of the time scale of the recovery of growth.     

INTERACTION OF POLY-L-LYSINE WITH CHROMATIN : Inhibition of In Situ RNA Synthesis Mediated by Escherichia coli RNA Polymerase

RNA polymerase from Escherichia coli was used in conjunction with labeled nucleosides as an autoradiographic reagent to study the availability of template in the chromatin of fixed nuclei and chromosomes Sequential treatments of the tissues with acid and poly-L-lysine were used to compare the effect of these treatments on the availability of template with the previously reported effects on the in situ priming for Escherichia coli DNA polymerase Acid treatment was found to increase the in situ activity of both enzymes, while poly-L-lysine strongly inhibited the in situ reactions mediated by RNA and DNA polymerases. When the DNA polymerase reaction was previously carried out on alcohol-fixed chicken blood smears, leukocyte nuclei primed extensively for DNA synthesis. In contrast, we did not detect incorporation into intact nuclei of any cell type in alcohol-fixed blood smears that were treated with RNA polymerase.     

Cytoplasmic steps of peptidoglycan synthesis in Escherichia coli.

The cellular pool levels of most of the cytoplasmic precursors of peptidoglycan synthesis were determined for normally growing cells of Escherichia coli K-12. In particular, a convenient method for analyzing the uridine nucleotide precursor contents was developed by associating gel filtration and reverse-phase high-pressure liquid chromatography techniques. The enzymatic parameters of the four synthetases which catalyze the stepwise addition of L-alanine, D-glutamic acid, meso-diaminopimelic acid, and D-alanyl-D-alanine to uridine diphosphate-N-acetylmuramic acid were determined. It was noteworthy that the pool levels of L-alanine, D-glutamic acid, meso-diaminopimelic acid, and D-alanyl-D-alanine were much higher than the Km values determined for these substrates, whereas the molar concentrations of the uridine nucleotide precursors were lower than or about the same order of magnitude as the corresponding Km values. Taking into consideration the data obtained, an attempt was made to compare the in vitro activities of the D-glutamic acid, meso-diaminopimelic acid, and D-alanyl-D-alanine adding enzymes with their in vivo functioning, expressed by the amounts of peptidoglycan synthesized. The results also suggested that these adding activities were not in excess in the cell under normal growth conditions, but their amounts appeared adjusted to the requirements of peptidoglycan synthesis. Under the different in vitro conditions considered, only low levels of L-alanine adding activity were observed.     

DEAD-box RNA helicases in Escherichia coli

In spite of their importance in RNA metabolism, the function of DExD/H-box proteins (including DEAD-box proteins) is poorly understood at the molecular level. Here, we present recent progress achieved with the five DEAD-box proteins from Escherichia coli, which have been particularly well studied. These proteins, which have orthologues in many bacteria, participate, in particular, in specific steps of mRNA decay and ribosome assembly. In vitro, they behave as poorly processive RNA helicases, presumably because they only unwind a few base pairs at each cycle so that stable duplexes can reanneal rather than dissociate. Except for one of them (DbpA), these proteins lack RNA specificity in vitro, and specificity in vivo is likely conferred by partners that target them to defined substrates. Interestingly, at least one of them is multifunctional, presumably because it can interact with different partners. Altogether, several aspects of the information gathered with these proteins have become paradigms for our understanding of DEAD-box proteins in general.     

DNA-Dependent RNA Polymerase Activity Associated with Subviral Particles of Polyhedral Cytoplasmic Dexoyribovirus

Polyhedral cytoplasmic deoxyribovirus virions contain a DNA-dependent RNA polymerase which catalyzes the incorporation of ribonucleotides into an acid-precipitable product. Treatment of virions with sodium deoxycholate and dithiothreitol resulted in the formation of subviral particles which could be separated from virions by rate zonal centrifugation in sucrose gradients. Subviral particles were RNA polymerase-positive and more active per unit mass of protein than virions. In vitro enzyme activity associated with subviral particles required addition of ribonucleotides, Mg(2+), and exogenous denatured DNA template. Optimal enzyme activity occurred over a broad pH (7.2 to 8.8) and Mg(2+) concentration (2 to 10 mumol) range. The specific activity of the RNA polymerase was maximal at 37 C. Addition of DNase or actinomycin D to the reaction mixture reduced the incorporation of [(3)H]UMP into an acid-precipitable product. The product of the reaction was sensitive to degradation by RNase but not to DNase or Pronase. These data suggest that the enzyme copies DNA into RNA.     

Comparison of Turnip Crinkle Virus RNA-Dependent RNA Polymerase Preparations Expressed in Escherichia coli or Derived from Infected Plants

Turnip crinkle virus (TCV) is a small, plus-sense, single-stranded RNA virus of plants. A virus-coded protein, p88, which is required for replication has been expressed and purified from Escherichia coli. In vitro assays revealed that the recombinant p88 has an RNA-dependent RNA polymerase (RdRp) activity and can also bind to RNA. Deletion of the N-terminal region in p88 resulted in a more active RdRp, while further deletions abolished RdRp activity. Comparison of the E. coli-expressed p88, the N-terminal deletion mutant of p88, and a TCV RdRp preparation obtained from infected plants revealed that these preparations show remarkable similarities in RNA template recognition and usage. Both the recombinant and the plant TCV RdRp preparations are capable of de novo initiation on both plus- and minus-strand satC and satD templates, which are small parasitic RNAs associated with TCV infections. In addition, these RdRp preparations can efficiently recognize the related Tomato bushy stunt virus promoter sequences, including the minus- and plus-strand initiation promoters. Heterologous viral and artificial promoters are recognized poorly by the recombinant and the plant TCV RdRps. Further comparison of the single-component recombinant TCV RdRp and the multicomponent plant TCV RdRp will help dissect the functions of various components of the TCV replicase.