5' Cap methylation of homologous poly A(+) RNA by a RNA (guanine-7) methyltransferase from Neurospora crassa.

RNA (guanine-7) methyltransferase, partially purified from $\text{N.}\text{crassa}$ mycelia, catalyzed the transfer of the methyl group from S-adenosylmethionine to the 5′ terminus of both $\text{N.}\text{crassa}$ poly A(+) RNA and reovirus unmethylated mRNA. RNase T₂ digestion of the $\text{in}\text{vitro}$ methylated poly A(+) RNA from $\text{N.}\text{crassa}$ yielded the “cap” structures m ⁷G(5′)pppAp and m ⁷G(5′)pppGp in a ratio of 2:1 respectively. RNase T₂ digestion of the $\text{in}\text{vitro}$ methylated reovirus mRNA yielded m ⁷G(5′)pppGp exclusively. The absence of mRNA 2′-0-methyltransferase activity in the enzyme preparation is consistent with the absence of 2′-0-methylation in $\text{N.}\text{crassa}$ mRNA [Seidel, B. L. and Somberg, E. W. (1978) Arch. Biochem. Biophys. $\text{187}$, 108–112]. This is the first isolation of an eucaryotic, cellular RNA (guanine-7) methyltransferase that has been shown to methylate homologous substrate.     

Utilization of the guanylyltransferase and methyltransferases of vaccinia virus to modify and identify the 5′-terminals of heterologous RNA species

Enzymes extracted from purified vaccinia virus particles were used to catalyze the guanylylation (i.e. capping) and/or methylation of heterologous RNA species containing two or three phosphates or the structure m⁷G(5′)pppN at their 5′-terminals. This procedure provides a novel and specific method of labeling the 5′-terminals with $\text{[}{}^{\mathrm{\alpha -32}}\text{P]GTP}$ or S-adenosyl-[methyl-³H]methionine. Analysis of the RNAs of satellite tobacco necrosis virus and tobacco mosaic virus that were modified in this manner indicated that the original 5′-terminal sequences were (p)ppApGpPy and m⁷G(5′)pppGpU, respectively, which were enzymatically converted to m⁷G(5′)pppA^mpGpPy and m⁷G(5′)pppG^mpU.     

Translational control of the expression of the β subunit gene of E., coli RNA polymerase

Using the plasmid pNF1337 as template, a mRNA preparation has been obtained that directs the $\text{in}\text{,}\text{vitro}$ synthesis of fMet-Val, the N-terminal dipeptide of the β subunit of RNA polymerase. RNA polymerase holoenzyme specifically inhibits the mRNA-directed synthesis of fMet-Val showing that the autoregulation by RNA polymerase of β,β′ synthesis is at the level of translation. L factor ($\text{nusA}$ gene product) stimulates the synthesis of fMet-Val from a DNA template but not from mRNA. Rifampicin has no effect on the mRNA-directed synthesis of fMet-Val or the ability of RNA polymerase to inhibit fMet-Val synthesis.     

Site-directed mutagenesis: Generation of an extracistronic mutation in bacteriophage Qβ RNA

The preparation in vitro and chemical characterization of bacteriophage Qβ RNA with an extracistronic mutation, a G → A transition in the 16th position from the 3′-terminus, is described. The 5′-terminal region of the Qβ minus strand was synthesized in vitro up to position 14 (inclusive) by using ATP and GTP as the only substrates. The mutagenic nucleotide analog N⁴-hydroxyCMP was then incorporated into position 15 instead of CMP. The minus strand was completed with the four standard ribonucleoside triphosphates, purified and used as a template for the synthesis of plus strands. Of the plus strand product, 33% had a G → A transition in the 16th position from the 3′-end (which corresponds to position 15 of the minus strand), as shown by nucleotide sequence analysis of the terminal T₁ oligonucleotide. The modified RNA was efficiently replicated by Qβ replicase and a preparation containing 55% of the mutant RNA was obtained.The general approach to directed mutagenesis outlined above should allow the introduction of mutations into the 5′ and 3′-terminal regions of Qβ RNA as well as into the intercistronic sequences.     

Cordycepin and xylosyladenine: inhibitors of methylation of nuclear RNA.

Cordycepin and xylosyladenine inhibited methylation of nuclear RNA to a greater extent than RNA synthesis in L1210 cells $\text{in vitro}$. Inhibition of base methylation, 2′-0-methylation and 5′cap methylation was equal to, 2 to 5-fold greater and 1.5-fold greater, respectively than inhibition of RNA synthesis. Cordycepin was more potent than xylosyladenine in inhibiting 2′-0-methylation of cytidine and adenosine, but not guanosine. These results suggest that impirment in the 2′-0-methylation of nuclear RNA may be one of the major effects that limits the biological activity of rRNA and mRNA by these drugs.     

Subunits of RNA polymerase in function and structure: VI. Sequence of the assembly in vitro of Escherichia coli RNA polymerase

Detailed analysis of the assembly in vitro of Escherichia coli RNA polymerase reveals that core enzyme subunits are assembled in the following sequence: $\text{2 α → α}{}_2\text{→}\text{β}\text{α}{}_2\text{β}\text{→}\text{β′}\text{α}{}_2\text{ββ′}$(premature core) → E (active core). Activation of the premature core enzyme, the rate-limiting step in this sequence, can be achieved in three different ways: self-reactivation, sigma subunit (σ or σ′)-promoted reactivation, and DNA-promoted reactivation.Although there has been disagreement on the enhancement of core enzyme maturation by sigma subunit or DNA, the discrepancy is resolved by the present finding that the premature core alone can be activated in the presence of high concentrations of salt or glycerol, whereas at a salt concentration as low as that in vivo, sigma subunit or DNA is required for maximum activation. However, the question remains unsolved as to which of the three ways operates in the in vivo process of RNA polymerase formation.     

RNA-protein interactions in the ribosome. I. Characterization and ribonuclease digestion of 16 S RNA-ribosomal protein complexes

Proteins from the 30 S ribosomal subunit of Escherichia coli were fractionated by column chromatography and individually incubated with 16 S ribosomal RNA. Stable and specific complexes were formed between proteins S4, S7, S8, S15 and S20, and the 16 S RNA. Protein S13 and one or both proteins of the $\text{S}\text{16}\text{S}\text{17}$ mixture bound more weakly to the RNA, although these interactions too were apparently specific. The binding of $\text{S}\text{16}\text{S}\text{17}$ was found to be markedly stimulated by proteins S4, S8, S15 and S20. Limited digestion of the RNA-protein complexes with T₁ or pancreatic ribonucleases yielded a variety of partially overlapping RNA fragments, which retained one or more of the proteins. Since similar fragments were recovered when 16 S RNA alone was digested under the same conditions, their stability could not be accounted for by the presence of bound protein. The integrity of the fragments was, however, strongly influenced by the magnesium ion concentration at which ribonuclease digestion was carried out. Each of the RNA fragments was characterized by fingerprinting and positioned within the sequence of the 1600-nucleotide 16 S RNA molecule. The location of ribosomal protein binding sites was delimited by the pattern of fragments to which a given protein bound. The binding sites for proteins S4, S8, S15, S20 and, possibly, S13 and $\text{S}\text{16}\text{S}\text{17}$ as well, lie within the 5′-terminal half of the 16 S RNA molecule. In particular, the S4 binding site was localized to the first 500 nucleotides of this sequence while that for S15 lies within a 140-nucleotide sequence starting about 600 nucleotides from the 5′-terminus. The binding site for the protein S7 lies between 900 and 1500 nucleotides from the 5′-terminus of the ribosomal RNA.     

Characterization of Neurospora crassa polyadenylated messenger ribonucleic acid: structure of the 5'-terminus.

Polyadenylated (poly(A)⁺) mRNA from Neurospora crassa was isolated by affinity chromatography on poly(U) Sepharose and its structure was examined. Two 5′-terminal ·cap’ structures, m⁷G(5′)ppp(5′)Ap and m⁷G(5′)ppp(5′)Gp, occurring in a relative distribution of 75 and 25% were found. No evidence was obtained for 2′-O-methylation in a nucleotide adjacent to the 5′-terminal cap.     

Effect of guanine nucleotides on the assembly of brain microtubles: ability of 5'-guanylyl imidodiphosphate to replace GTB in promoting the polymerization of microtubules in vitro.

Polymerization of microtubule requires the presence of GTP, and the tubulin-bound GTP is hydrolyzed during microtubule formation. However, it was found that an unhydrolyzable analog of GTP, 5′-guanylyl imidodiphosphate (Gpp(NH)p), was able to replace GTP. The hydrolysis of the terminal phosphate group of GTP, therefore, does not seem to be a prerequisite to $\text{in vitro}$ assembly of microtubules. The microtubules formed in the presence of Gpp(NH)p were indistinguishable from those formed in the presence of GTP under electron microscopy, but a remarkable decrease was noted in their sensitivity to depolymerization by calcium ions.     

Structure analysis of three T7 late mRNA ribosome binding sites

The 5′-terminal regions of the three T7 late RNA species IIIb, IV and V have been characterized. These regions contain the protein synthesis initiation sites for the T7 genes 17, 9 and 10, respectively. Each of these is located between 60 and 90 nucleotides from the 5′ terminus of an in vitro synthesized RNA species. The sequence 5′ A-C-U-U-U-A-A-G-Pu-A-G-Pu, which is common to these ribosome binding regions, contains an impressive stretch of complementarity to the sequence 5′ A-C-C-U-C-C-U-U-A, at the 3′ terminus of 16 S ribosomal RNA. The nuclease mapping technique of Wurst et al. (1978) has been used to probe intramolecular structural interactions involving these initiation regions in the RNA. My results indicate that all three initiation codons, together with other portions of the ribosome binding regions are protected, under non-denaturing conditions, against the actions of both the single-strand-specific nuclease S₁ and RNAase T₁.     

Preparation and stability of the helical form of poly 2'-O-ethyluridylic acid

2′ (3′)-O-ethyl-CMP was prepared by alkylation of CMP with diethylsulphate in alkaline medium and deaminated to give 2′(3′)-O-ethyl-UMP, which was phosphorylated to 2′(3′)-O-ethyl-UDP. About 90% of the product consisted of the 2′ isomer. The 2′(3′)-O-ethyl-UDP was readily polymerized by $\text{E}$. $\text{coli}$ polynucleotide phosphorylase in the presence of Mn⁺⁺, but not Mg⁺⁺. The 3′-isomer did not seriously interfere with polymerization nor did it act as a chain terminator. The resulting poly 2′-O-ethyluridylic acid formed a helical structure with a stability much higher then that of poly (rU) or poly 2′-O-methyluridylic acid. It also complexed readily with poly (rA). Implications with regard to the role of the 2′-hydroxyl in nucleic acid conformation are discussed.     

Inhibition of noncapped mRNA translation by the cap analogue, 7-methylguanosine-5'-phosphate.

The cap analogue, 7-methylguanosine-5′-phosphate (pm⁷G), inhibits the translation of the noncapped STNV (satellite tobacco necrosis virus) RNA and CPMV (cowpea mosaic virus) RNA in the $\text{in vitro}$ wheat germ protein synthesizing system. While the translation of some capped mRNAs is inhibited more strongly by the analogue, other capped mRNAs have a level of sensitivity similar to that of the noncapped RNAs. Evidence is presented demonstrating that the effect of the analogue is exerted at a cap binding site even when it is inhibiting noncapped mRNAs. These results therefore indicate that the cap binding site of the translational system is either part of or is closely linked to another mRNA binding component, this component being specific for a site on the mRNA other than the 5′ cap. The observations also suggest caution in the use of pm⁷G inhibition to indicate the presence of a 5′ cap on a particular mRNA.