An Introduction to Molecular Biotechnology. Группа авторов
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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.
References
1 Voet, D., Voet, J.G., and Pratt, C.W. (2002). Fundamentals of Biochemistry, upgrade edition. New York: Wiley.
2 Voet, D., Voet, J.G., and Pratt, C.W. (2016). Fundamentals of Biochemistry, Live at the Molecular Level, 5e. New Jersey: Wiley.
Further Reading
1 Alberts, B., Johnson, A., Lewis, L. et al. (2015). Molecular Biology of the Cell, 6e. New York: Garland Science.
2 Alberts, B., Bray, D., Hopkin, K. et al. (2019). Essential Cell Biology, 5e. New York: Garland Science.
3 Krebs, J., Goldstein, E.S., and Kilpatrick, S.T. (2018). Lewin's Genes XII. Burlington: Jones & Bartlett Learning.
3 Structure and Functions of a Cell
Michael Wink
Heidelberg University, Institute of Pharmacy and Molecular Biotechnology (IPMB), Im Neuenheimer Feld 329, 69120 Heidelberg, Germany
3.1 Structure of a Eukaryotic Cell
3.1.1 Structure and Function of the Cytoplasmic Membrane
The hydrophilic or hydrophobic interactions of many lipid molecules in the aqueous cell environment give rise to the spontaneous formation of energetically favorable membrane bilayers. These are fluid, plastic, and mobile (Figures 2.2 and 3.1). Although the individual phospholipids spin around themselves and constantly move laterally, the resulting membrane is not easily permeable for ions, and charged or polar molecules.
Figure 3.1 Mobility of phospholipids in a biomembrane. Three types of movement are possible: rotation (spin), lateral diffusion, and flip‐flop, which occurs rarely. A flip‐flop can be brought about with the enzyme flippase.
Under cellular conditions, biomembranes tend not to lie flat like a carpet, but assume a spherical shape (Figure 3.2a). Should holes and ruptures in the cytoplasmic membrane occur, they are only transient and immediately resealed. These remarkable self‐organization and formation of supramolecular structures were prerequisites for the emergence of cells – and thus of life itself. Membranes can easily invert to form vesicles that, in turn, can merge with other membranes. When a vesicle is pinched off from a biomembrane, this is called exocytosis. When a vesicle is absorbed by a compartment membrane, it is called endocytosis.