NUCLEIC ACID STRUCTURE III

Tertiary Structure

Ribonucleic Acids (RNA)

RNA molecules are smaller than DNA molecules in any particular organism. RNA molecules can be single-stranded or double-stranded. RNA molecules can be linear or circular (but no circular, double-stranded RNA molecules have been described). The most abundant RNA in cells is ribosomal RNA (rRNA).

Many of the original standard nucleotides in tRNAs are altered into modified bases by post-transcriptional enzymatic action (Figure 27.7). There are 86 tRNAs in E. coli. Most tRNAs are about 75-80 nucleotides long and have extensive secondary structure (base pairing interactions) (Figure 27.6) as well as tertiary structure (not supercoiling as we will discuss below, but additional folding in three dimensional space). All tRNAs end with ...CCA at the 3'-end. Amino acids are attached to the 3'-ends of tRNAs to form aminoacyl-tRNAs (charged tRNAs). Figure E1 shows examples of various types of unusual base pairing found in DNA and RNA. Figure E2 shows the stablization of tRNA secondary and tertiary structure by base-base interactions. In addition to Watson-Crick base pairs accounting for most of the interactions (especially in the stem regions), there are a number of nonstandard base pairs and base triplets.

Supercoiling

Covalently closed, circular, double-stranded DNA molecules can be supercoiled. This corresponds to the tertiary structure of DNA; that is, the higher order folding of elements of secondary structure. Negative supercoiling is underwinding (has more base pairs/turn). Positive supercoiling is overwinding (has less base pairs/turn). Otherwise identical molecules that differ only in their state of supercoiling are called topoisomers = topological isomers.

Figure 24.29 shows that topoisomers can identified by gel electrophoresis. DNA is visualized by ultraviolet light after staining with ethidium bromide. DNA is negatively charged and migrates towards the positive pole. Breaking even one phosphodiester bond (a nick) of a supercoiled DNA molecule converts it into a relaxed circle. Enzymes, called topoisomerases (see below), can catalyze changes in the linking number of covalently closed circular DNA molecules (in steps of 1 by type I topoisomerases and in steps of 2 by type II topoisomerases).

Figure E3 shows that supercoiling is characterized by the linking number (Lk), the twist (Tw), and the writhe (Wr), where

Tw = twist = number of turns of the double helix

Wr = writhe = number of superhelical turns; Wr is positive for positive supercoiling and negative for negative supercoiling

Lk = linking number = Tw + Wr

We often are only interested in the change of linking number: Lk = Tw + Wr

Another index of supercoiling is the superhelical density = = Wr/Tw. Superhelical density gives the number of supercoils for each helical turn of the DNA. Naturally occurring DNA molecules in prokaryotic and eukaryotic cells are typically negatively supercoiled with = -0.06.

Note: Two or more interlinked circular DNA molecules have quaternary structure. This would be the equivalent of proteins having two or more subunits. Interlinked circular DNA molecules could also independently differ in their linking number. Interlinked circles occurs at the termination of replication of circular DNA molecules (such as the E. coli chromosome). Interlinked circles can be decatenated by type II topoisomerases, especially E. coli topoisomerase IV (see below).

Do you know supercoiling? Test yourself!

Eukaryotic DNA is complexed with basic proteins called histones to form chromatin. Table 28.1 lists the properties of histones. Histone assemble as a specific octamer containing two copies each of H2A, H2B, H3, and H4. DNA wraps around the histone octamer in a left-handed direction to form a nucleosome. Figure 28.11 digrams the elements of chromatin structure. Experimentally, the repetitive nucleosomal structure of chromatin is revealed by nuclease digestion; Figure 28.8 shows the 200 base pair nucleosome repeat pattern after treatment of chicken erythrocyte chromatin with micrococcal nuclease. Figure E4 shows that wrapping of DNA around the histone octamer creates negatively supercoiled DNA. The X-ray crystal structure of the nucleosome core particle has been determined. Modifying chromatin structure during DNA replication and RNA synthesis (transcription) requires elaborate machinery.

Topoisomerases:

Type IA family (change linking number in steps of 1)

E. coli topoisomerase I:
Requires magnesium. Can cleave DNA or RNA. Binds preferentially to the junctions between double- and single-stranded regions of DNA. Covalent intermediate between a tyrosine and a 5'-phosphate at the cleavage site. Can only relax negatively supercoiled molecules: requires a region of single-strandedness (when topo I binds to negatively supercoiled DNA, the binding energy is sufficiently large to overcome the unfavorable free-energy of unpairing a short stretch of the DNA helix; the binding energy with positively supercoiled DNA is not sufficient to unwind the helix).

Type IB family (change linking number in steps of 1)

Eukaryotic topoisomerases I (human, mouse, yeast, etc.):
Does not requires metal cofactor. Does not need a stretch of single-stranded DNA; therefore, can relax both positively and negatively supercoiled DNA molecules. Forms a covalent intermediate between a tyrosine and a 3'-phosphate at the cleavage site.

Type II family (change linking number in steps of 2)

E. coli topoisomerase II, also known as gyrase:
A multimer enzyme (two subunits encoded by gyrA and gyrB genes). About 140 bp of DNA wraps around the enzyme in a right-handed sense accounting for preferential binding to positively supercoiled DNA. Forms an intermediate between tyrosine and 3'-phosphate at cleave sites. Can relax both positive and negative supercoiling. Can actively introduce negative supercoils in an ATP-dependent manner. Relatively poor at catenation/decatenation and knotting/unknotting of DNA circles. This enzyme acts as the SWIVEL for DNA replication.

E. coli topoisomerase IV:
A multimer enzyme (parC and parE genes encode the two subunits), CANNOT catalyze negative supercoiling, but is efficient at catenation/decatenation and knotting/unknotting of DNA circles in an ATP-dependent manner. Can relax both positive and negative supercoiling. This enzyme is required to unlink the two daughter DNA molecules at the conclusion of DNA replication.

Eukarotic topoisomerases II (human, mouse, yeast, etc.). Has preference for catenation/decatenation and knotting/unknotting of DNA circles like E. coli topoisomerase IV.