7.1 Introduction Until now, we have discussed DNA primarily as a double helix, but in its natural state within the cell it is found packaged as a complex mixture with many different proteins and other components. You have already seen examples of proteins with specific roles to play, such as topoisomerases and the proteins with various DNA binding domains, but in this section we will turn our attention to the proteins that serve to pack and organise the DNA into what we call chromatin. The packaging of
6.1 Introduction The biological functions of both DNA and RNA are dependent on complex, and sometimes transient, three-dimensional nucleoprotein structures. It is in such structures that the enzymatic manipulation of DNA in the essential biological processes such as DNA replication, transcription and recombination occur, and it is important to understand the interactions that drive these processes. Whether it is the activity of enzymes associated with DNA, such as DNA topoisomerases, or the packaging of DNA o
Alkylating agents There are many different chemicals that directly chemically modify DNA. The methyl group (—CH3) can be added to DNA at various sites, two of which we will discuss further. There are several alkylation-sensitive sites in guanosine (identified in Table 3e). For example, methylation at the 7-position of the guanine base (
The loss of a DNA base causes an abasic site Hydrolysis of the deoxyribose Cl'–base linkage results in the complete loss of a purine or pyrimidine base, resulting in what is called an abasic site, an event with obvious genetic consequences. This hydrolysis reaction is much more likely to occur at purine bases, resulting in depurination of the DNA (Table 3a<
4.6 Summary RNA chains play fundamentally important roles within the cell, including genetic information transfer (mRNA), components of the translation machinery (rRNA in ribosomes and tRNAs) and as regulatory small RNAs. The tertiary structure of RNA is determined by interactions that maximise base pairing. Despite instability and isolation problems, the tertiary structures of several major cellular RNAs are known. Transfer RNA struct
Aptamers Aptamers are nucleic acid molecules that have been developed to mimic the selective and tight binding of other molecules such as antibodies. In order to identify an aptamer that is capable of binding to a target molecule, a process called Selex (systematic evolution of ligands by exponential enrichment) is utilised. The strategy relies upon a combination of a selective binding assay and amplification by PCR. A ‘library’ of short single-stranded DNA oligonucleotides is synthesised <
Antisense regulation of gene expression The term antisense refers to the use of a nucleic acid that is complementary to the coding (i.e. ‘sense’) base sequence of a target gene. When nucleic acids that are antisense in nature are introduced into cells, they can hybridise to the complementary ‘sense’ mRNA through normal Watson-Crick base pairing. Synthetic antisense DNA chains as short as 15–17 nucleotides in length have been used to block specific gene expression by either physically blocking translation of the tar
4.3 Hairpin formation and micro-RNAs A class of small RNA molecules called micro-RNAs (miRNAs) has been identified in recent years. The roles of these small RNAs are only just beginning to be understood, but many are expressed only at specific developmental stages. Indeed, the first observations of miRNAs were made in C. elegans because of their mutant developmental phenotypes. The genes that encode these miRNAs are called mir genes (pronounced ‘meer’) and have now been identified within the genomes of v
4.2 The structure of tRNA Transfer RNAs are small and compact molecules. Comparisons of the base sequences of many tRNAs led to the predicted four-leaf clover structure shown in Figure 18a, which follows the rule of maximising base-pairing interactions. This structure was largely confirmed by analysis with single-strand nucleases. Two of the four main arms of the tRNA molecule are named according to their function, i.e. binding to the mRNA trinucleotide that encodes a specific amino acid (anticodon arm),
4.1 The varied structures of RNA RNA is a versatile cellular molecule with the ability to adopt a number of complex structural conformations. Although RNA is often thought of as a single-stranded molecule it is actually highly structured. Summary of Section 3 Watson–Crick base pairing arises due to hydrogen bonding between A and T and G and C and spatial limitations within the hydrophobic core of the helix. DNA commonly folds into the B-form helix; other forms such as Z-DNA form in vitro. A-form helices are formed primarily by duplex RNA. The twisting of DNA around its helical axis results in torsional stresses that promote the formation of high-energy alternative confo Triplex structures An unusual form of three-stranded structure, called triplex DNA, can arise in vitro when a single-stranded region of DNA pairs with a paired duplex DNA helix through additional hydrogen bonding between the bases of all three strands. The fluidity of torsional stress along the DNA chain The fluid changes in conformation and free energy of the DNA helix are influenced by many processes including the binding of proteins, some of which may have a regulatory function. Thus binding of a protein in one position along a DNA chain could result in alterations in the topology of the DNA, and hence changes in free energy availability, both locally and at some distance from the binding site. Changes in torsional energy may serve as an indicator of the state of the surrounding helix. For Torsional energy can be taken up by alternative DNA conformations The energy introduced into DNA by twisting has great potential as a regulatory mechanism, since the free energy can be stored in a variety of different high-energy conformations along the chain. 3.1 The helical structure of DNA Having outlined the general principles of nucleic acid structures, we will now focus on how these principles influence the formation of specific structures found in DNA. The helical structure of DNA arises because of the specific interactions between bases and the non-specific hydrophobic effects described earlier. Its structure is also determined through its active synthesis; that is, duplex DNA is synthesised by specialist polymerases upon a template strand. Within the helix, the two Summary of Section 2 Nucleic acids are intrinsically highly flexible molecules. The chemical properties of nucleic acid components are primary determinants in structure formation. The formation of nucleic acid structures is driven by base pairing and stacking interactions between the hydrophobic bases. In DNA, these interactions drive the formation of the double helix, whose structure is maintained under torsional stress by twisting. RNA second 2.4 Analysis of nucleic acids by electrophoresis and hybridisation Nucleic acids can be separated according to size by gel electrophoresis, most commonly performed using a horizontal gel (Figure 7a). This is in contrast to the vertical gel electrophoresis set-up, which is generally used for analysis of proteins. The size of DNA molecules is usually expressed in terms of the number of 2.3 Analysing nucleic acid structures In studying nucleic acid structures, many different experimental approaches can be adopted. In many cases, nucleic acid structures are examined in vitro, under non-physiological conditions, such as after denaturation or chemical synthesis. Nucleic acids within a cell are formed under very specific conditions and the structures that they adopt are influenced not only by the nature of their synthesis (by DNA or RNA polymerases), but by ancillary proteins that influence their folding. Nev Base pairing Nucleic acid folding patterns are dominated by base pairing, which results from the formation of hydrogen bonds between pairs of nucleotides. In nucleic acids, as in proteins, the highly directional nature of this hydrogen bonding is the key to secondary structure. 2.2 General features of higher-order nucleic acid structure Polynucleotide chains are intrinsically flexible molecules and have the potential to form many different higher-order structures. Their flexibility derives from rotation around bonds in the sugar-phosphate backbone (Figure 3b). In vivo, the structures that form are obviously determined by both the proteins that synthesise the nucleic acid chains (polymerases) and the ancillary proteins that bind to and modify them. We will discuss these aspects of structure later in this unit. What dri
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