Читать книгу An Introduction to Molecular Biotechnology - Группа авторов - Страница 17

Nucleotides play important roles in the cell: as energy carriers (ATP, adenosine diphosphate [ADP]); as coenzymes (FAD, NAD⁺, coenzyme A), during the transfer of sugar moieties (ADP‐glucose); and as building blocks for nucleic acids (Figure 2.17a). Nucleotides consist of the purine bases adenine and guanine and the pyrimidine bases cytosine and thymine or uracil, which form N‐glycosidic bonds with ribose or deoxyribose. The 5′‐hydroxyl group of the pentose is esterified with one, two, or three phosphate residues (Figure 2.17b).

Figure 2.17 Structure of nucleotides. (a) Structures of purine and pyrimidine bases, pentoses, and ATP (as an example of a nucleotide). (b) Structures of ATP, AMP, ADP, glucose, FAD⁺, and coenzyme A.

Our genetic information is stored in the form of deoxyribonucleic acid(DNA). DNA is a macromolecule and is made up of nucleotide subunits bound together linearly (Figure 2.18). DNA contains the bases A, T, G, and C; RNA contains the bases A, U, G, and C. The nomenclature of the bases, nucleosides, and nucleotides is explained in Table 2.7.

Figure 2.18 Linear structure of DNA and RNA. In nucleic acid biosynthesis, the α‐positioned phosphate group of a nucleotide triphosphate (NTPs in RNA, dNTPs in DNA) is linked to the free 3′‐OH group of the available strand.

The nucleotides are the building blocks for DNA and RNA. Nucleotides are esterified into polynucleotide chains via a phosphate backbone. The 5′‐hydroxyl group (“five prime hydroxyl group”) of a pentose is linked via a phosphodiester bond to the 3′‐hydroxyl group of a second pentose (Figure 2.18). During the biosynthesis of the nucleic acids, the respective nucleotide triphosphates are needed whose phosphoric acid anhydride bonds are especially rich in energy. In the completed nucleic acid, only nucleotide monophosphates are present. After cleavage of a diphosphate residue, the α‐phosphate group attacks the free 3′‐end of the already existing nucleic acid strand and forms a new ester bond. The synthesis is said to occur in the 5′ → 3′ direction.

DNA exists as a double helix whereby the bases A and T, and G and T, respectively, face each other in a complementary manner (Figure 2.19). Both DNA strands are arranged antiparallel to each other (i.e. within a helix one of the strands runs in the 5′ → 3′ direction, while the complementary partner strand is oriented in the 3′ → 5′ direction). The DNA double helix has a diameter of 2 nm.

Figure 2.19 Structure of the DNA double helix. The spatial orientation of the base pairs in the double helix and the principle of complementary base pairing between A and T, and G and C, respectively, via the formation of hydrogen bonds. (a) Schematic structure of the double helix. (b) Structural formula.

Complementary base pairing is achieved through the specific formation of two or three hydrogen bonds between A–T and G–C pairs, respectively (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; T_m(melting temperature) indicates the temperature at which 50% of the DNA is already present as single strands. T_m is dependent on the GC content of the DNA, which varies significantly between organisms. The higher the GC content, the higher the average T_m (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 Sons.

(B) Secondary structure of 16S rRNA. a. Schematic representation, b. Structure of rRNA of the large ribomsome subunit of bacteria; colours of domains are identical to those in B.a; c. front view.

Source: Courtesy of V. Ramakrishan, MRC, Cambridge.

(C) An example of 23S rRNA (from Haloarcula marismortui, a halophilic red Archaeon found in the Dead Sea) with six domains (Domain I‐VI). a. Schematoic view; bv. Tertiary structure with six domains.

Source: Courtesy of Thomas Steitz and Peter Moore, Yale University.

RNA interference(RNAi) describes a widely distributed phenomenon in which double‐stranded RNA molecules lead to the breakdown of complementary mRNA. In the cell there is a ribonuclease (so‐called Dicer), which can cleave the double‐stranded RNA into short, 21‐ to 23‐nucleotide siRNA(short interfering RNA) molecules. The siRNA assembles itself together with proteins and forms the RNA‐induced silencing complex(RISC), which binds to the mRNA that is complementary to siRNA (e.g. of viruses or transposons). By cleaving the mRNA, the associated gene activity is inhibited. SiRNA regulates gene expression and rearrangements, by switching off transposons.

A further group of small noncoding RNA molecules are the miRNAs (micro‐RNAs). An endogenous single‐stranded RNA molecule is produced by RNA polymerase II, which is then trimmed to miRNA 21–23 nucleotides in length by Dicer. miRNAs have been found in plants and animals. miRNA binds and inactivates complementary mRNA molecules and seems to play a very important role in gene regulation, differentiation, and tissue development.

The RNAi method is an important tool for basic research in order to examine the function of genes. By introducing double‐stranded siRNA through transfection or with the help of a particle gun, targeted inhibition of gene activity is possible. It is also possible to produce transgenic cells that produce siRNA themselves. siRNA is a further development of the antisense RNAs and plays an important role as a tool for cellular/molecular biology and developmental biology, in order to silence all the genes of an organism in a specific way. Biotechnologists are also working on developing these molecules as therapeutics.

Also the CRISPR‐mediated immunity of bacteria against viral infections employs small noncoding RNAs (crRNAs), similar to miRNA and siRNAs. When bacteria become infected with a virus, they manage to integrate short viral sequences into their genomes. These viral sequences become the templates for crRNAs, which can detect a future viral pathogen. When crRNAs have detected a complementary viral RNA, the latter is degraded by CRISPR‐associated proteins (Cas). The CRISPR/Cas system has recently been developed into a powerful system for gene editing in plants and animals, which avoids the traditional problems of recombinant DNA.

Catalytically active RNA molecules are important in ribosomes and were supposedly present in early evolution. These RNAs were surrounded by a simple biological membrane. They contained the genetic information and were also responsible for structure formation and catalysis. In addition to other tasks, they carried out protein synthesis. It is assumed that there was a division of labor further in the course of evolution, so that DNA took over the storage of genetic information and proteins took over the role as catalysts and structure carriers. Today, RNA has important roles both as a messenger between DNA and protein, as well as a catalytic and regulatory molecule.

Ribozymes are short RNA molecules that recognize and specifically cleave their target RNA via shared base sequences (Figure 2.21). Through selection of new ribozymes, biotechnologists are attempting to develop new enzyme‐like catalysts or therapeutics that can switch off unwanted gene activity.

Figure 2.21 Structure and function of a hammerhead ribozyme.