RNase folding

The studies of RNase Aand cytochrome c (Qi etal., 1998 Sosnick etal., 1997) show that caution is required in interpreting burst phenomena in protein folding. However, they do not require a reinterpretation of the cases in which the molten globule character of the burst-intermediate has been established (Arai and Kuwajima, 2000 Chamberlain and Marqusee, 2000). 

An example of this effect is provided by ribonuclease A (RNase A). At pH 8 and 37°, the rate of deamidation of Asn67 was more than 30-fold lower in the native than in the unfolded protein [111]. Deamidation of the native RNase A was also ca. 30-fold slower than of an octapeptide whose sequence is similar to that of the deamidation site, although the reaction mechanisms were similar [108][123],... 

Studies of proteolytic fragments of staphylococcal nuclease (Tan-iuchi and Anfinsen, 1969) and RNase A (Taniuchi, 1970) seemed to support this view. Taniuchi (1970), in summary remarks, said Thus, the minimum information of the specific folding of a protein requiring almost the entire amino acid sequence is observed with both staph-yloccocal nuclease and bovine pancreatic ribonuclease. ... 

Many secretory proteins—e. g., pancreatic ribonuclease (RNAse see p. 74)—contain several disulfide bonds that are only formed oxidatively from SH groups after translation. The eight cysteine residues of the RNAse can in principle form 105 different pairings, but only the combination of the four disulfide bonds shown on p. 75 provides active enzyme. Incorrect pairings can block further folding or lead to unstable or insoluble conformations. The enzyme protein disulfide iso-merase [1] accelerates the equilibration between paired and unpaired cysteine residues, so that incorrect pairs can be quickly split before the protein finds its final conformation. 

The catalytic core domain of HIV-1 integrase has a topologically identical fold with the RNase H domain of HIV-1 reverse transcriptase [37], the RuvC Holli-... 

Structures of the catalytic core of HIV-1 integrase, HIV-l RNase H, RuvC, and the core domain of MuA transposase demonstrating similarities in folding topology. The catalytically essential residues... 

RGURE 8-27 Base-paired helical structures in an RNA. Shown here is the possible secondary structure of the M1 RNA component of the enzyme RNase P of coli, with many hairpins RNase R which also contains a protein component (not shown), functions in the processing of transfer RNAs (see Rg. 26-23). The two brackets indicate additional complementary sequences that may be paired in the three-dimensional structure. The blue dots indicate non-Watson-Crick G=U base pairs (boxed inset). Note that G=U base pairs are allowed only when presynthesized strands of RNA fold up or anneal with each other. There are no RNA polymerases (the enzymes that synthesize RNAs on a DNA template) that insert a U opposite a template G, or vice versa, during RNA synthesis... 

Figure 28-8 Simplified scheme for control of replication of the ColEl type plasmid by antisense RNA. The primer for DNA synthesis is RNA II whose initial transcript extends past the replication ori. It is cut by RNase H at ori and then primes replication of the upper strand as shown in the figure. The antisense RNA is RNA I. It hinds to protein Rop whose gene location is also indicated in the figure. Rop assists RNA I and RNA II in undergoing a complementary interaction. However, both RNAs apparently maintain a folded tertiary structure, and only some segments interact. The interaction with the Rop protein evidently in some way prevents initiation of replication until the Rop concentration falls because of replication of the host cell.167,168...

The rate of hydrolysis of DNA, RNA, and polynucleotides can be measured by a sensitive spectrophotometric assay which is based on the hyperchromicity that occurs upon hydrolysis of these substrates (S). The enzyme has a 7-fold greater affinity for denatured DNA than for RNA (8). No inhibitory products accumulate during the course of the reaction. The pH optimum for RNase and DNase activities is between 9 and 10, depending on the Ca2+ concentration. At higher pH values less Ca2+ is required. The inhibitory effect of high Ca2+ observed consistently by many investigators is more pronounced at higher pH values (S). 

When guanosine 2, 3 -cyclic phosphate is incubated with about 3-fold nucleoside at a low temperature in the presence of RNase N, guanylyl-(3, 5 )-nucleoside can be obtained as a synthetic product. For example, using uridine as a phosphate acceptor, GpU was obtained with a high yield of 27% calculated upon guanosine 2, 3 -cyclic phosphate added. This... 

Furthermore, when ApG cyclic-p was incubated with about 14-fold cytidine at a low temperature in the presence of RNase N, trinucleoside diphosphate, ApGpC could be obtained with the yield of about 38% after 1 hr of incubation. When ApG cyclic-p alone was incubated with RNase N, the polymerized products with the definite repeating sequence (ApGp)n could be produced. The yield of tetramer (n = 2), ApGpApG cyclic-p -f- ApGpApGp, amounted to 34% and that of hexa-mer (n = 3), to 7%. But larger polymers were scarcely detected (T. Koike, T. Uchida, and F. Egami, unpublished). 

An enzyme similar to the 3 -nucleotidase of mung bean has been isolated from germinating wheat seedlings and purified 800-fold (90). The preparation possessed DNase, RNase, and 3 -nucleotidase activities. These three activities were similar in pH optima, requirements for Zn2+ and sulfhydryl compounds, stability to storage, temperature inactivation... 

The E. coli chromosome exists as a circular, folded, supercoiled duplex (a). This can be converted to a partially unfolded structure by brief treatment with RNase (c). There are 50-100 loops in the structure supercoiling may be selectively eliminated from individual loops by single-strand nicking of the DNA within the loop (b). 

Proteins that bind to the RNA may influence the folding. As a consequence, patterns of sequence co-variations that have evolved for RNAs that are functional in complexes with proteins (e. g., rRNA, RNAse P-RNA) might not conform very well with predicted folding for the isolated RNA. 

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