rel="nofollow" href="#ulink_ea33887f-1b27-5721-9ee5-46c03d86f0a8">Figure 2.19). This is an important example of a molecular recognition reaction via noncovalent bonds. Base pairing occurs spontaneously should the two bases meet. This results in the ability to self‐organize and to form supramolecular structures without the requirement of energy or regulatory helpers. The selectivity of complementary base pairing is an important requirement for basic genetic processes (e.g. replication, transcription, and recombination) and diagnostic procedures (e.g. Southern hybridization, DNA fingerprinting with DNA probes, quantitative PCR, and DNA microchips; see Chapters 21, 22, and 27).
In eukaryotes, the multiple negative charges on the backbone of the DNA double helix are complexed with basic, positively charged histone proteins (Figure 4.6); in prokaryotes, positively charged polyamines take over this role. The bases are arranged inside of the helix and form planar stacks (Figure 2.19). The inside of the helix is anhydrous – only lipophilic substances, especially if they are also planar, can be inserted in between the base stacks (so‐called DNA intercalators). Such intercalation often leads to errors during replication, which can initiate frameshift mutations and strand breaks (see Section 4.1.5).
Determined by the cooperativity of many hydrogen bonds and the lipophilic interactions between the base stacks, the DNA double helix is very stable and can only be separated into the single strands by high temperatures. This process is also called melting; Tm(melting temperature) indicates the temperature at which 50% of the DNA is already present as single strands. Tm is dependent on the GC content of the DNA, which varies significantly between organisms. The higher the GC content, the higher the average Tm (caused by three hydrogen bonds in G–C pairs vs. two hydrogen bonds in A–T pairs); this is practically important when primers or DNA probes are to be designed. If these primers/probes are to be hybridized under stringent conditions, primers with a higher GC content are preferred.
Important enzymes that use DNA as their substrate are summarized in Table 2.8. Many of these enzymes are important tools in molecular biology and biotechnology (see Chapter 12).
Table 2.8 Enzymes that use DNA as a substrate and are used in genetic engineering.
| Enzyme | Reaction |
|---|---|
| Restriction endonuclease | Cuts DNA at specific palindromic recognition sequences that are 4–6 bp long |
| DNA polymerase I | Synthesis of the complementary DNA strand; requires a primer with a free 3′‐end; important for DNA sequencing |
| DNA ligase | ligates (joins together) DNA strands; the enzyme forms phosphodiester bonds between neighboring phosphate residues |
| Telomerase | Synthesizes telomere sequences at the end of chromosomes |
| DNA topoisomerases | Cuts DNA strands, either single or double stranded |
| Taq polymerase | Heat‐stable DNA polymerase from Thermus aquaticus; important for PCR |
| DNase | Hydrolase that cleaves double‐stranded DNA |
| RNase | Hydrolase that degrades single‐ or double‐stranded RNA |
| RNA polymerase | Copies DNA into mRNA and rRNA |
| Reverse transcriptase | Copies RNA into DNA |
As opposed to DNA, the RNA world is much more complex. The basic structure of RNA, from the four ribonucleotides A, U, G, and C, is valid for all RNA species. RNA molecules initially occur as single strands. As partial sequences within an RNA molecule are often complementary, RNA double strands form spontaneously (so‐called stem structures). Nonpaired regions form single‐stranded loop structures. RNA can interact with several diverse molecules via the nonpaired bases and can be catalytically active (e.g. by formation of peptide bonds in ribosomal protein biosynthesis or the splicing of nucleic acids).
RNA often exhibits characteristic structures and functions (Figure 2.20):
mRNA. Messenger RNA codes for proteins; in eukaryotes with a cap structure on the 5′‐end and a poly(A) tail on the 3′‐end.
tRNA. Transfer RNA, adaptor between mRNA and amino acids; with posttranscriptional base modifications in loop regions.
rRNA. 5S, 23S, and 16S rRNA in prokaryotic ribosomes with characteristic secondary and tertiary structures.
rRNA. 5S, 5,8S, 18S, and 28S rRNA in eukaryotic ribosomes with characteristic secondary and tertiary structures. Catalyze protein synthesis.
snRNA. Small nuclear RNA; catalyzes pre‐mRNA splicing.
snoRNA. Small nucleolar RNA; chemically modify rRNA.
siRNA. Small interfering RNA; small double‐stranded RNA molecules that can influence gene expression by directing degradation of selective mRNAs and the establishment of compact chromatin structures.
miRNA. microRNA; small single‐strand RNA molecules that can control gene activity, development, and differentiation by specifically blocking translation of particular mRNA.
piRNA. Piwi‐interacting RNAs; bind to Piwi proteins and protect germline from transposable elements.
lncRNA. Long noncoding RNAs are conserved in genomes; they apparently play a role in regulating gene transcription.
Ribozymes. RNA with catalytic activity.
Figure 2.20 Structure of RNA molecules. (A) Yeast tRNA. The base sequence is described as clover shaped. The thin lines depict the tertiary interactions between the base pairs. The bases circled in solid lines are those that are conserved in all tRNAs. Those bases circled in dotted lines are only semiconserved.
Source: Voet et al. (2002). Reproduced with permission of John Wiley and
