Nucleotide sequences of the 5' termini of phi X174 mRNAs synthesized in vitro

When using X174 RFI DNA as a template, in vitro, E. coli RNA polymerase synthesizes four major purine triphosphate-containing 5′ end sequences. RNase A digests of α³²P labeled RNA were further digested with spleen exonuclease to remove the bulk of the oligonucleotides with 5′ hydroxyls and then chromatographed on DEAE cellulose to resolve the remaining 5′ terminal oligonucleotides. By application of standard separation and sequence techniques, the major 5′ end sequences were shown to be: pppApUp(Cp), pppApApApUp(Cp), pppApApApApUp(Cp), and pppGpApUp(Gp).     

Nucleotide sequence of the cloned gene for bacteriophage T7 RNA polymerase

The coding sequence of the gene for bacteriophage T7 RNA polymerase has been cloned into the PstI site of plasmid pBR322. This cloned DNA extends from T7 nucleotide (Dunn & Studier, 1981) 3127 to 5821 or T7 coordinate 7.82 to 14.55. The nucleotide sequence is given, as well as the predicted amino acid sequence of T7 RNA polymerase. This peptide sequence is comprised of 883 amino acid residues with a total molecular weight of 98,092.     

Early to late switch in bacteriophage T7 development: functional decay of T7 early messenger RNA

Infection of ultraviolet light-irradiated Escherichia coli with T7 phage in the presence of chloramphenicol results in synthesis of T7 early messenger RNA but not late mRNA. T7 early mRNA accumulates in terms of acid-insoluble, T7 DNA-hybridizable RNA. However, messenger activity of the same RNA decays rapidly with a half-life of about 6.5 minutes at 30 °C when tested for the ability to direct in vitro protein synthesis. This functional decay of T7 early mRNA is attributable to a loss of structural integrity of the RNA. Polyacrylamide-agarose gel electrophoresis shows that T7 early mRNAs are cleaved, generating smaller-size RNAs. Kinetics of the appearance of T7-specific RNA polymerase, one of the early gene products, during normal T7 infection show that the capacity of the cells to produce the enzyme decays very rapidly when early mRNA synthesis is terminated either by rifampicin or by a natural mechanism programmed by T7. Preferential synthesis of late proteins in the presence of chemically stable early mRNA late in T7 infection may be explained by the observed functional decay of early mRNA.     

Nucleotide sequence of the three major early promoters of bacteriophage T7.

I have determined the nucleotide sequences of the three major early promoters of bacteriophage T7 (A1, A2, A3). The sequences confirm the two main homologies found between other known promoters for E. coli RNA polymerase (nucleoside triphosphate:RNA nucleotidyl transferase, E.C. 2. 7. 7. 6). In particular, all three T7 promoters show a very good match with the -35 region homology; the A2 and A3 promoters share a 17 basepair sequence in this region. On the other hand, the match with the Pribnow Box homology is much less pronounced and different for each T7 promoter.     

Effect of ribosomal loading on the structural stability of bacteriophage T7 early messenger RNAs.

The $\text{in vivo}$ structural stabilities of the T7 early mRANs were measured and found to vary according to whether chloramphenicol or puromycin were added before or after infection with phage T7. These antibiotics had little effect upon messenger stability when they were added prior to infection. When chloramphenicol (but not puromycin) was added after completion of T7 early mRNA synthesis, the structural stability of the messages was enhanced. Messages which are inefficiently translated $\text{in vivo}$ due to altered 5′-termini were not stabilized by the late addition of chloramphenicol. We interpret these results to mean that ribosomal protection of the T7 early mRNAs is responsible for the increase in messenger structural stability in the presence of chloramphenicol.