The translation efficiency of ovalbumin mRNA is determined in part by a 5'-end hairpin structure.

We have investigated the role in translation of the secondary structure in the noncoding leader (NCL) sequence of ovalbumin mRNA. Deletion of a stem-loop structure from the mRNA 5'-end (hairpin-1) produced a 2.5-fold decrease in mRNA translation rate in both rabbit reticulocyte wheat germ cell-free systems. A corresponding 2-fold reduction in mRNA binding affinity for reticulocyte eucaryotic initiation factor 2 (eIF-2) was also observed. These effects were independent of mRNA capping. Both translation rate and eIF-2 binding affinity were restored by addition to the mRNA of a sequence containing a hairpin-1-like structure. The positive correlation between cell free translation rate and mRNA binding to eIF-2 suggests that hairpin-1 is both an initiation signal and part of a specific eIF-2 recognition site. Methylation interference indicated a direct interaction between eIF-2 and hairpin-1 of ovalbumin mRNA.

The 5'-end structure of ovalbumin mRNA in isolated nuclei and polysomes.

We used the chemical reagents dimethylsulfate and 4'-aminomethyl-4,5',8-trimethylpsoralen and the enzyme T1 ribonuclease to compare the 5'-end structure of ovalbumin mRNA in situ in purified hen oviduct nuclei and polysomes with that of the isolated mRNA. The qualitative pattern of structure-dependent base modifications and T1 ribonuclease cleavage sites in intranuclear and polysomal ovalbumin mRNAs was found to be nearly identical to those in isolated ovalbumin mRNA. These structural data are consistent with the presence of a trigonal stem-loop structure at the 5'-end of ovalbumin mRNA (hairpin-1) in nuclei and polysomes. Similar results were obtained for a coding region structure (hairpin-3) in intranuclear ovalbumin mRNA. We have recently shown that hairpin-1 positively affects the rate of ovalbumin mRNA translation in vitro and is part of a high affinity binding site for eucaryotic initiation factor-2 (eIF-2). The presence of hairpin-1 in ovalbumin mRNA in both a pretranslation state (nuclei) and active translation state (polysomes) is consistent with its hypothesized biological function as an intracellular initiation signal that facilitates the translation of this mRNA.

A simple assay for determining the capping efficiencies of RNA polymerases used for in vitro transcription.

We have developed an assay that uses phenyl boronate agarose column chromatography to measure the capping efficiencies of RNA polymerases used for in vitro transcription of cloned cDNAs. Capped 32P-labeled ovalbumin mRNAs were synthesized by in vitro run-off transcription with SP6 or T7 RNA polymerase in the presence of cap analogs and digested to completion with T1 and T2 RNase. The resulting 3'-nucleoside monophosphates (NMPs) and cap structures were separated by chromatography on phenyl boronate agarose, and the ratio of radioactivity between the two was used to estimate the extent of transcript capping.

Determination of secondary structure in the initiation region of ovalbumin mRNA.

We have analyzed the secondary structure in the region surrounding the initiation codons of both cellular and synthetic versions of ovalbumin mRNA. RNase V1 cleavage sites and structure-dependent, chemically modified bases in cellular ovalbumin mRNA were determined by reverse transcription of hen poly A(+) RNA using ovalbumin-specific, synthetic DNA primers. These results indicate an extensive region of unpaired nucleotides preceding the initiation codon and a region of base-paired nucleotides including and following the initiation codon. A synthetic ovalbumin mRNA (SP65.OV) was prepared by run-off transcription of a cloned ovalbumin cDNA (pSP65.OV). Identical regions of hen ovalbumin and SP65.OV mRNAs gave identical patterns of structure-dependent base modifications. A computer program for determining RNA secondary structure was used to find a 5'-region structure for ovalbumin mRNA that is consistent with our data.

Characterization of the effects on ovalbumin mRNA of aminomethyl-trimethylpsoralen photoreaction with hen oviduct mRNA.

We have described the reaction of 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) with hen oviduct mRNA and have investigated the specific effects of AMT photoaddition on ovalbumin mRNA which constitutes 60-70% of oviduct mRNA. The photoreaction of AMT with hen oviduct mRNA appeared to occur in two phases - a rapid monoaddition followed by a slower conversion of monoadducts to diadducts (i.e. crosslinking). Both nondenaturing and denaturing gel electrophoresis revealed a photoreaction time dependent increase in ovalbumin mRNA electrophoretic mobility indicating the formation of a progressively more compact molecular structure. Identical analysis of photoreacted rabbit globin mRNA revealed no change in electrophoretic mobility suggesting that AMT was stabilizing the AU rich secondary structure of ovalbumin mRNA but having no similar effect on the relatively GC rich secondary structure of globin mRNA. Ovalbumin-specific DNA primer extension was used to demonstrate the selective and secondary structure specific photoaddition of AMT to uracil bases at the 5' end of ovalbumin mRNA.

Identification of the major immunoglobulin heavy chain poly A+ RNA in murine lymphoid tissue.

Total cellular Poly A+ RNA from TEPC15 myeloma and murine lymphoid tissues was analyzed by denaturing agarose gel electrophoresis and Northern blot hybridization to specific heavy chain constant region cDNAs; mu, gamma 2b and alpha RNA species were identified in each of the tissues and in the IgA producing TEPC15 myeloma. In total Poly A+ RNA from TEPC15 myeloma, alpha-cDNA hybridized predominantly to a 2.3 kb RNA species; 11.5 and 4.1 kb RNA species were evident as well. Successive hybridization of the same RNA to mu- and gamma 2b-specific cDNAs demonstrated the presence of both mu and gamma 2b specific RNA species with electrophoretic mobilities apparently identical to the 11.5 and 2.3 kb RNA species identified by alpha-specific hybridization. These data establish the presence of Poly A+ RNA species containing alpha, mu, and gamma 2b sequences in TEPC15 cells and suggest the transcription of RNA from both CH-containing chromosomes in TEPC15 myeloma (one chromosome in the TEPC15 cell line contains all the CH genes while the other chromosome has deleted all CH genes except alpha). In total Poly A+ RNA from normal mouse tissues (Peyer's patch and spleen) all three cDNA probes hybridized predominantly to an 11.5 kb RNA species. Primer extension experiments demonstrated that alpha cDNA could prime for the synthesis by reverse transcriptase of gamma 2b DNA when Peyer's patch Poly A+ RNA was used as the template. This suggests the existence of a single transcript containing alpha and gamma 2b sequences. Murine lymphoid tissues contained putative mRNAs for mu, gamma 2b and alpha heavy chain proteins, whereas TEPC15 myeloma polysomal Poly A+ RNA contained only alpha mRNA.

Factors influencing the covalent binding of (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene to ribonucleic acids.

The covalent binding of the ultimate carcinogen (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo [alpha]pyrene (BPDE) to enriched ovalbumin messenger RNA (mRNAov) of known sequence was examined. Incubation of mRNAov with elevated concentrations of labeled BPDE in TE buffer (0.02 M Tris X HCl, 1 mM EDTA, pH 7.2) containing 0.1 M KCl and 10 mM MgCl2 resulted in approximately 30 BPDEs covalently bound per RNA molecule. Covalent binding in the absence of KCl and MgCl2 resulted in a significant increase in binding to 110 BPDEs bound per molecule or modification of 12% of the total guanosine and adenosine nucleotides present. The nucleoside adducts formed were nearly all guanosine and adenosine in a ratio of 1.6:1.0. It was also observed that digestion of mRNAov with T2 RNase prior to reaction with BPDE resulted in a 52% decrease in guanosine adduct formation and a 93% decrease in adenosine adducts compared with undigested controls. Comparison of the binding of labeled BPDE to 18 S and 28 S ribosomal RNAs and to mRNAov revealed that the guanosine adduct to adenosine adduct ratio and the number of BPDEs bound increased with increasing G-C content. The results reported here show that ionic composition of the medium, G-C content, and the presence of a polymeric state can significantly influence the quantitative and/or qualitative nucleoside BPDE adducts formed.

Nonspecific cleavage of phi X174 RFI deoxyribonucleic acid by bleomycin.

The covalently closed circular duplex deoxyribonucleic acid (DNA) of phi X174 underwent progressive conversion to nicked and linear DNA with increasing bleomycin/phi X174 RFI DNA molecule ratios. The formation of linear DNA (a double-strand break) occurred under limited reaction conditions as low as an average of 0.2 single-strand break/phi X174 RFI DNA molecule. As bleomycin-produced linear DNA was further fragmented by bleomycin, a broad distribution of DNA fragments without notable concentrations of unique size was formed. Restriction enzymes PstI and SstII did not generate discrete fragments from bleomycin-produced full-length linear phi X174 DNA, nor did bleomycin cleavage generate discrete fragments from HpaII or PstI digests of phi X174 RFI. These findings suggest that bleomycin does not act at a few specific sites on phi X174 RFI DNA. The single-strand nick appeared to be the preferred site for bleomycin action for a second cleavage in a phi X174 molecule.

Ribosome binding site analysis of ovalbumin messenger ribonucleic acid.

The region of the ovalbumin messenger ribonucleic acid (mRNAov) molecule bound to the 40S ribosomal subunit and its associated initiation factors in the wheat germ cell-free translation system were isolated and characterized. Two mRNAov fragments, 87 and 92 nucleotides in length, were protected from T1 ribonuclease digestion by binding of guanosine 5',beta,gamma-methylenetriphosphate and were shown by hybridization and fingerprint mapping to be derived from the 5' end of mRNAov. Both these mRNAov fragments were of sufficient length to contain both the cap structure and the AUG initiation codon. Four T1-resistant oligonucleotides, prepared by direct digestion of mRNAov with T1 ribonuclease were also found to bind to the wheat germ 40S ribosomal subunit. Nucleotide sequence analysis of these oligonucleotides revealed (1) that they were not a subset of the ribosome binding fragments described above, (2) that they were derived from within the mRNAov molecule (one from within the coding region and three from the noncoding region at the 3' end of the mRNAov molecule), and (3) that three of the four mRNAov nucleotides contained 3'-terminal AUG trinucleotides. These data suggested that features of the mRNAov molecule in addition to the nucleotide sequence might be important in specifying the correct ribosome binding site for the initiation of protein synthesis. The amount of mRNAov bound to the wheat germ 40S ribosomal subunit in a preinitiation complex was found to vary inversely with the potassium ion concentration. Lowering the potassium concentration to levels suboptimal for translation also resulted in the protection of larger fragments of the mRNAov molecule derived from the same 5'-end region as the ribosome binding fragments described above. The ability of the cap analogue 7-methylguanosine 5'-phosphate (m7G5'p) to reduce the amount of mRNAov bound to the wheat germ 40S ribosomal subunit was found to depend directly on thepotassium concentration. Interestingly, the effects of potassium on the amount of mRNAov bound in a preinitiation complex and the inhibition of this binding by m7G5'p could be observed by changing the potassium concentration after binding had occurred. These data suggested that the interaction between the wheat germ 40S ribosomal subunit and mRNAov was very sensitive to the ionic environment.

Nucleotide sequence at the 5' end of ovalbumin messenger RNA from chicken.

DNA-sequence analysis of 300 nucleotides from the region of cloned, double-stranded ovalbumin cDNA corresponding to the 5' end of ovalbumin messenger RNA was accomplished using the technique of Maxam and Gilbert (Proc. Nat. Acad. Sci. USA (1977) 74,560-564). The AUG initiation codon was located 52 nucleotides from the AT linkers used in cloning and immediately adjacent to the amino terminal peptide of ovalbumin, indicating the absence of a "signal peptide" in this protein. The nucleotide sequence coding for a phosphorylated peptide from ovalbumin was also found. These results demonstrate that the coding portion of mRNAov begins near the 5' end of the molecule leaving some 600 nucleotides of noncoding information at the 3' end.

Physical and chemical characterization of purified ovalbumin messenger RNA.

Preparative agarose gel electrophoresis under denaturing conditions has been successfully employed to purify large quantities of ovalbumin mRNA from hen oviducts. The mRNA thus prepared is physically homogeneous based on its migration as a single component on electrophoresis in both analytical acid-urea agarose gels and formamide-containing, neutral polyacrylaminde gels; it also sediments as a single peak in sucrose gradients containing 70% formamide. The mRNA is chemically free of ribosomal RNA contamination since its oligonucleotide fingerprint map after complete T1 ribonuclease digestion contains no detectable specific large oligonucleotide markers of ribosomal RNAs. It is also not contaminated by other biologically active messenger RNAs because, when it is added to the cell-free wheat germ translation system, the only protein product synthesized is ovalbumin as analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and specific immunoprecipitation. Ovalbumin mRNA has a nucleotide composition of 32.3% A, 21.0% G, 25.7% U, and 20.7% C [(A+U)/(G+C) equal 1.41]. The mRNA contains a heterogeneous poly(A) tract ranging from 20 to 140 residues with a number average chain length of 62 adenylate residues. The molecular weight of the sodium salt of the purified mRNA is approximately 650,000 +/- 63,000, corresponding to a chain length of 1890 +/- 180 nucleotides, as determined by electron microscopy under completely denaturing conditions. This value is in close agreement with the values obtained from: (a) sucrose gradient centrifugation in the presence of 70% formamide; (b) evaluation of poly(A) content in the mRNA and the number average chain length of its poly(A) tract; and (c) sedimentation velocity studies in the presence of 3% formaldehyde. When 125I-labeled ovalbumin mRNA is allowed to hybridize with a large excess of chick DNA, the observed kinetics of hybridization reveal no appreciable reaction between the mRNA and the repeated sequences of the chick DNA, although the mRNA appears to be approximately 600 nucleotides longer than necessary to code for ovalbumin. It thus appears that the entire ovalbumin mRNA is primarily transcribed from a unique sequence in the chick genome.