Structural Studies on Intact and Deadenylylated Rabbit Globin mRNA

Specific hydrolysis of rabbit globin messenger RNA by S1 nuclease

“…The influence of salt concentration and temperature on the SI digestion is consistent with changes in helical content in globin mRNA observed using optical methods (25,27), and is entirely analogous to results obtained with single strand nuclease digestions of SV40 DNA (14,15), MS2 RNA (13) and tRNA (17,34). The data reported here, along with our previous observations (25), show that agents known to disrupt helical structure (high temperature, low salt, formaldehyde) increase SI susceptibility. The results indicate that S1 nuclease can be used as a probe for specif ic doub le stranded structure in mRNA.…”

“…SI nuclease activity is maximal at pH 4.0-4.3, with almost no activity above pH 6.0 (12), necessitating the use of an acidic condition for these studies. Estimates of secondary structure at neutral pH range from 52-70% (25,27,32). The larger amount of nuclease resistance that we find is not unreasonable, since helical structure in globin mRNA increases as the pH is lowered (25).…”

“…The percentage of nucleotides protected from the single strand specific nuclease is rather high and varies depending on the SI concentration. Similarly, high estimates of helical structure in globin mRNA were obtained at pH 4.5 by thermal denaturation (25). SI nuclease activity is maximal at pH 4.0-4.3, with almost no activity above pH 6.0 (12), necessitating the use of an acidic condition for these studies.…”

“…SI nuclease was purified from crude ot-amylase powder by the method of Vogt (12). The single strand specificity was confirmed using the synthetic polynucleotides 3H-poly(U) and 3H-poly(A), as previously described (25). Enzyme activity was determined according to the method of Vogt (12), using sonicated, heat denatured, calf thymus DNA as substrate.…”

The role of mRNA structure in the regulation of protein synthesis: Implications for studies of development

“…Translation of mRNA can be affected by factors that affect RNA structure such as ionic conditions [ 181. Direct measurements by physical techniques [19][20][21][22], and enzymatic means [23,24] demonstrate that eukaryotic mRNA has considerable specific secondary structure. The experiments of Rosa [25] using enzymatic mapping techniques developed in this laboratory defined the secondary structures of initiation regions in three T7 phage late mRNA species, IIIb, IV and V. Although prokaryotic ribosomes locate the correct initiator through base-pairing between the 3'-end of 16s rRNA and the region immediately preceding the AUG, the factors that affect the translational efficiency of an mRNA are unclear.…”