RNAs and Their Constituents

This technique has widely been applied in a series of papers by Clementi and coworkers for the description of the solvation of amino acids, peptides as well as of RNA, DNA and their constituents (for reviews see 161 l62>). The interactions of some cations with these types of molecules were also described 163 166>. Pair potentials between small model molecules and the cations Li+ 62), Na+ 167), K+ l68), NII4+ I68), Mg2+ 98> and Ca2 + 98) were developed in order to describe ion-ionophore interactions169>. 

The nomenclature of nucleotides and their constituent units was presented earlier (Section 5.1.2). Recall that a nucleoside consists of a purine or pyrimidine base linked to a sugar and that a nucleotide is a phosphate ester of a nucleoside. The names of the major bases of RNA and DNA, and of their nucleoside and nucleotide derivatives, are... 

Figure 3.14 Structures of common nucleosides whose acid-catalysed hydrolysis has been studied. Adenosine, guanosine and cytidine are three of the four common nucleosides in RNA and their 2 -deoxy derivatives in DNA, whereas uridine is found only in RNA and 2 -deox5hh5midine in DNA. Psicofuranine is an antibiotic and is not a common constituent of nucleic acids. Inosine is a commonly used substrate in investigations of enzymic ribosyl transfer.

Ribosomes are composed of two basic subunits— the 70S ribosome being composed of a 50S and a 30S subunit. These have been further fractionated into their RNA and protein constituents and in some cases have been reassembled to form active ribosomes. Omission of various components during reassembly allows investigation of the functions of the omitted component and it has been shown, for example, that one of the ribosomal proteins is involved in the... 

Both DNA and RNA are easily broken down by acid-catalyzed hydrolysis. Tlius, heating at 100°C for one hour in 12 M HC104 is sufficient to hydrolyze nucleic acids to their constituent bases. However, for analysis of RNA it is better to heat in 1 N HC1 for 1 h at... 

Composition of the E. coli ribosomes. The 70S ribosome can dissociate into a 50S and a 30S subunit. In vitro this can be done by lowering the Mg ion concentration. The individual subunits can be dissociated into their constituent RNAs and proteins by exposure to urea denaturant. Molecular weights are given for the subunits and the proteins, and the numbers of nucleotides are given for the RNAs. 

Nucleic acid structures also involve assembly events determined by differential solubilities of different molecular constituents. The stacking of purine and pyrimidine bases in the helical structures of DNA relies on hydrophobic effects, whereas the positioning of phosphate groups in contact with solvent reflects their hydrophilic nature. Secondary structures of RNA likewise are influenced by differential solubilities of polar and nonpolar constituents. 

Many other examples of outwardly complex molecular structures, whose salient architectural features appear to self-assemble from their constituent building blocks, have been documented [16]. The formation of the DNA double helix from its constituent chains is perhaps the quintessential example, whilst the perfect reconstitution of the intact tobacco mosaic virus from its constituent RNA and protein monomers also exhibits all the hallmarks of a cooperative self-assembly process [17]. The same is true of ribonuclease. Reconstitution of this enzyme in the presence of mercaptoethanol, to allow reversible exchange of the four disulfide bridges, proceeds smoothly to generate eventually only the active conformation from many possible isomeric states [18], In each of these cases, the thermodynamic stability of the product is vital in directing its synthesis. These syntheses could therefore be termed product-directed. 

As mentioned, many other molecules exist in the saliva, including nucleic acids (RNA, DNA), several hormones, growth factors and neurotransmitters, amino acids and their derivatives, urea, lactate, citrate, vitamins, creatinine, prostaglandins, several drugs, and chemical constituents of foods, cosmetics, tooth pastes, dental materials, and several other molecules originated from body and environment. 

A ribosome can be dissociated into a large subunit (50S) and a small subunit (SOS) (Figure 29.15). These subunits can be further split into their constituent proteins and RNAs. The SOS subunit contains 21 different proteins (referred to as SI through S21) and a 16S RNA molecule. The 50S subunit contains 34 different proteins (LI through L34) and two RNA molecules, a 23S and a 5S species. A ribosome contains one copy of each RNA molecule, two copies of the L7 and L12 proteins, and one copy of each of the other proteins. The L7 protein is identical with L12 except that its amino terminus is acetylated. Only one protein is common to both subunits S20 is identical with L26. Both the SOS and the 50S subunits can be reconstituted in vitro from their constituent proteins and RNA, as was first achieved by Masayasu Nomura in 1968. This reconstitution is an outstanding example of the principle that supramolecular complexes can form spontaneously from their macromolecular constituents. 

Enzymes are proteins that catalyze biochemical reactions. A catalyst is a substance that greatly accelerates the rate of a particular reaction without being used up or permanently altered- In the real world, most catalysts eventually deteriorate and no longer function as a catalyst. In the cell, all enzymes are eventually degraded and converted back to their constituent amino acids plus, in some cases, byproducts of oxidation or other types of damage- Ptoteins do not have some unique magical property that allows them to function as enzymes. For certain activities nucleic acids also participate in the chemistry of catalysis- For example, mRNAcan catalyze certain types of RNA splicing. 

Nucleic acid molecules absorb ultraviolet light maximally at 260 nra owing almost entirely to the constituent bases. Thus DNA or RNA yield can be quantified by spectrophotomet-ric measurement of the absorbance at 260 nm. Alternatively, isolated nucleic acids can be subjected to agarose gel electrophoresis and their yield quantified by densitometric measurements. 

A ribosome can be dissociated into a large subunit (5OS) and a small sub-unit (30S). These subunits can be further split into their constituent proteins and RNAs. The 30S subunit contains 21 different proteins (referred to as Si through S21) and a 16S RNA molecule. The SOS subunit contains 34 different proteins (LI through L34) and two RNA molecules, a 23S and a. 55... 

DNA and RNA can be radiolabeled with carbon-14,hydrogen-3,or phosphorus-32 and then separated into their constituent nucleotide sequences by enzymatic digestion. Restriction enzymes cut double-stranded DNA through each of the sugar-phosphate backbones, without damaging the nucleotide bases. 

Unlike DNA, RNA is disposable Many copies of an RNA sequence are made from a single DNA sequence. These copies are used and recycled back to their constituent nucleotides. This allows the cell to respond quickly to changing conditions by transcribing different sequences into RNA. Special sequences called promoters tell RNA polymerase, the enzyme responsible for transcription, where to start making RNA (Figure 4-4). 

Nucleic acids absorb strongly in the near-UV region because of the purine and pyrimidine bases they contain. However, the energies of the strong transitions for the five most common bases are virtually the same, which results in very close absorbance maxima (260, 246, 259, 265, and 267nm for adenine, guanine, uracil, thymine and cytosine, respectively), even though their exact position is pH dependent. This makes it rather difficult to assess the contribution of each band to the spectra of DNA or RNA, which exhibit a broad absorbance band between 240 and 280 nm due to the bases. The spectrophotometric quantification of nucleic acids is also hindered by marked variations in the spectra with pH as a result of the ionization of their constituent bases. 

Ribosomal RNA is the most abundant form of RNA and forms about 80% of the total. In combination with numerous proteins it forms the ribosome that is the work bench on which proteins are assembled from their constituent amino acids. The ribosome consists of two subunits. In eukaryotes a 40S subunit combines with a 60S subunit to form an SOS ribosome (Fig. 69.1). Prokaryotes are different, they have a 30S subunit that combines with a SOS subunit to form a 70S ribosome (Fig. 69.1). 

In view of the need to understand these new effects and their implications for helix and loop formations in RNA and DNA the conformational ramifications of the constituent dimers themselves should be sorted out as thorou ly as possible. Notwithstanding the large number of studies that have appeared recently (vide infra), general agreement on the thermodynamics and geometry of helix formation has not been reached. 

That uridine or cytidine can be the exclusive precursor of the pyrimidine deoxyribonucleotide subunits of DNA was shown by Karlstrdm and Larsson ( 9) with a pyrimidine-requiring E. coli mutant, OK305. After growing the bacteria in the presence of uridine or cytidine totally labeled with C, DNA and RNA were isolated and degraded to their constituent nucleotides. The specific activities of the sugar and base portions of the nucleotides from RNA and DNA were compared (Tables 14r-I and 14-11). 

In experiments of this type with animal tissues, Rose and Schweigert (2) and Thomson et al. (S) showed that pyrimidine ribonucleosides were converted to DNA nucleotides without cleavage of the JNT-glycosidic bond. As well, Larsson and Neilands (4) performed a similar type of experiment in which P-phosphate and uniformly labeled C-cytidine were administered to rats with regenerating liver. Both substances were incorporated into the liver polynucleotides which, upon isolation, were degraded to their constituent nucleotides for analysis. Their data (Table 16-1) showed that the four nucleotides of RNA had similar specific activities with respect to P, indicating that the labeled phosphate readily equilibrated with the nucleoside phosphate pool during the experimental period. In this experiment, cytidylate derived from RNA and deoxycytidylate derived from DNA had the same P C ratio. This result indicated that both polynucleotide subunits, deoxycytidylate and cytidylate, were derived from a common precursor, evidently a ribonucleotide. 

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