H. Lodish - Molecular Cell Biology (5ed, Freeman, 2003) (796244), страница 45
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(a) Electron micrograph ofSV40 viral DNA. When the circular DNA ofthe SV40 virus is isolated and separated fromits associated protein, the DNA duplex isunderwound and assumes the supercoiledconfiguration. (b) If a supercoiled DNA isnicked (i.e., one strand cleaved), the strandscan rewind, leading to loss of a supercoil.Topoisomerase I catalyzes this reaction andalso reseals the broken ends. All the supercoilsin isolated SV40 DNA can be removed by thesequential action of this enzyme, producingthe relaxed-circle conformation. For clarity, theshapes of the molecules at the bottom havebeen simplified.Different Types of RNA Exhibit VariousConformations Related to Their FunctionsAs noted earlier, the primary structure of RNA is generallysimilar to that of DNA with two exceptions: the sugar component of RNA, ribose, has a hydroxyl group at the 2 position (see Figure 2-14b), and thymine in DNA is replaced byuracil in RNA.
The hydroxyl group on C2 of ribose makesRNA more chemically labile than DNA and provides achemically reactive group that takes part in RNA-mediatedcatalysis. As a result of this lability, RNA is cleaved intomononucleotides by alkaline solution, whereas DNA is not.Like DNA, RNA is a long polynucleotide that can be doublestranded or single-stranded, linear or circular. It can also participate in a hybrid helix composed of one RNA strand andone DNA strand. As noted above, RNA-RNA and RNADNA double helices have a compact conformation like the Aform of DNA (see Figure 4-4b).Unlike DNA, which exists primarily as a very long double helix, most cellular RNAs are single-stranded and exhibita variety of conformations (Figure 4-8).
Differences in thesizes and conformations of the various types of RNA permitthem to carry out specific functions in a cell. The simplestsecondary structures in single-stranded RNAs are formed bypairing of complementary bases. “Hairpins” are formed bypairing of bases within ≈5–10 nucleotides of each other, and“stem-loops” by pairing of bases that are separated by >10 toseveral hundred nucleotides.
These simple folds can cooperate to form more complicated tertiary structures, one ofwhich is termed a “pseudoknot.”As discussed in detail later, tRNA molecules adopt a welldefined three-dimensional architecture in solution that is crucial in protein synthesis. Larger rRNA molecules also havelocally well-defined three-dimensional structures, with moreflexible links in between. Secondary and tertiary structuresalso have been recognized in mRNA, particularly near theends of molecules. Clearly, then, RNA molecules are likeproteins in that they have structured domains connected byless structured, flexible stretches.The folded domains of RNA molecules not only arestructurally analogous to the helices and strands found inproteins, but in some cases also have catalytic capacities.Such catalytic RNAs are called ribozymes.
Although ribozymes usually are associated with proteins that stabilizethe ribozyme structure, it is the RNA that acts as a catalyst.Some ribozymes can catalyze splicing, a remarkable processin which an internal RNA sequence is cut and removed, andthe two resulting chains then ligated. This process occursduring formation of the majority of functional mRNA molecules in eukaryotic cells, and also occurs in bacteria and archaea. Remarkably, some RNAs carry out self-splicing, withthe catalytic activity residing in the sequence that is removed.The mechanisms of splicing and self-splicing are discussedin detail in Chapter 12.
As noted later in this chapter, rRNA108CHAPTER 4 • Basic Molecular Genetic Mechanisms FIGURE 4-8 RNA secondary(a) Secondary structureand tertiary structures. (a) Stem-loops,hairpins, and other secondary structurescan form by base pairing betweendistant complementary segments ofan RNA molecule. In stem-loops, thesingle-stranded loop between the basepaired helical stem may be hundredsor even thousands of nucleotides long,whereas in hairpins, the short turn maycontain as few as four nucleotides.(b) Pseudoknots, one type of RNAtertiary structure, are formed byinteraction of secondary loops throughbase pairing between complementarybases (green and blue). Only basepaired bases are shown. A secondarystructure diagram is shown at right.[Part (b) adapted from P.
J. A. Michiels et al.,2001, J. Mol. Biol. 310:1109.](b) Tertiary structure3Hairpin3Loop1Stem1Double-helicalstem region5Stem-loopplays a catalytic role in the formation of peptide bonds during protein synthesis.In this chapter, we focus on the functions of mRNA,tRNA, and rRNA in gene expression. In later chapters wewill encounter other RNAs, often associated with proteins,that participate in other cell functions.KEY CONCEPTS OF SECTION 4.1Structure of Nucleic AcidsDeoxyribonucleic acid (DNA), the genetic material, carries information to specify the amino acid sequences ofproteins.
It is transcribed into several types of ribonucleicacid (RNA), including messenger RNA (mRNA), transferRNA (tRNA), and ribosomal RNA (rRNA), which function in protein synthesis (see Figure 4-1).■Both DNA and RNA are long, unbranched polymers ofnucleotides, which consist of a phosphorylated pentoselinked to an organic base, either a purine or pyrimidine.■The purines adenine (A) and guanine (G) and the pyrimidine cytosine (C) are present in both DNA and RNA. Thepyrimidine thymine (T) present in DNA is replaced by thepyrimidine uracil (U) in RNA.■Stem2Loop25Pseudoknotside and the two sugar-phosphate backbones on the outside (see Figure 4-3).
Base pairing between the strands andhydrophobic interactions between adjacent bases in thesame strand stabilize this native structure.The bases in nucleic acids can interact via hydrogenbonds. The standard Watson-Crick base pairs are G·C, A·T(in DNA), and A·U (in RNA). Base pairing stabilizes thenative three-dimensional structures of DNA and RNA.■Binding of protein to DNA can deform its helical structure,causing local bending or unwinding of the DNA molecule.■Heat causes the DNA strands to separate (denature).The melting temperature Tm of DNA increases with thepercentage of G·C base pairs.
Under suitable conditions, separated complementary nucleic acid strands willrenature.■Circular DNA molecules can be twisted on themselves,forming supercoils (see Figure 4-7). Enzymes called topoisomerases can relieve torsional stress and remove supercoils from circular DNA molecules.■Cellular RNAs are single-stranded polynucleotides, someof which form well-defined secondary and tertiary structures (see Figure 4-8). Some RNAs, called ribozymes, havecatalytic activity.■Adjacent nucleotides in a polynucleotide are linked byphosphodiester bonds.
The entire strand has a chemical directionality: the 5 end with a free hydroxyl or phosphategroup on the 5 carbon of the sugar, and the 3 end witha free hydroxyl group on the 3 carbon of the sugar (seeFigure 4-2).4.2 Transcription of Protein-CodingGenes and Formation ofFunctional mRNANatural DNA (B DNA) contains two complementary antiparallel polynucleotide strands wound together into a regular right-handed double helix with the bases on the in-The simplest definition of a gene is a “unit of DNA that contains the information to specify synthesis of a single polypeptide chain or functional RNA (such as a tRNA).” The vast■■1094.2 • Transcription of Protein-Coding Genes and Formation of Functional mRNAmajority of genes carry information to build protein molecules, and it is the RNA copies of such protein-coding genesthat constitute the mRNA molecules of cells.
The DNAmolecules of small viruses contain only a few genes,whereas the single DNA molecule in each of the chromosomes of higher animals and plants may contain severalthousand genes.During synthesis of RNA, the four-base language of DNAcontaining A, G, C, and T is simply copied, or transcribed,into the four-base language of RNA, which is identical exceptthat U replaces T. In contrast, during protein synthesis thefour-base language of DNA and RNA is translated into the20–amino acid language of proteins. In this section we focuson formation of functional mRNAs from protein-codinggenes (see Figure 4-1, step 1 ).
A similar process yields theprecursors of rRNAs and tRNAs encoded by rRNA andtRNA genes; these precursors are then further modified toyield functional rRNAs and tRNAs (Chapter 12).A Template DNA Strand Is Transcribed intoa Complementary RNA Chain by RNA PolymeraseDuring transcription of DNA, one DNA strand acts as a template, determining the order in which ribonucleoside triphosphate (rNTP) monomers are polymerized to form acomplementary RNA chain. Bases in the template DNAstrand base-pair with complementary incoming rNTPs,which then are joined in a polymerization reaction catalyzedby RNA polymerase. Polymerization involves a nucleophilicattack by the 3 oxygen in the growing RNA chain on the phosphate of the next nucleotide precursor to be added, resulting in formation of a phosphodiester bond and releaseof pyrophosphate (PPi). As a consequence of this mechanism,RNA molecules are always synthesized in the 5n3 direction (Figure 4-9).The energetics of the polymerization reaction strongly favors addition of ribonucleotides to the growing RNA chainbecause the high-energy bond between the and phosphate of rNTP monomers is replaced by the lower-energyphosphodiester bond between nucleotides.
The equilibriumfor the reaction is driven further toward chain elongation bypyrophosphatase, an enzyme that catalyzes cleavage of thereleased PPi into two molecules of inorganic phosphate. Likethe two strands in DNA, the template DNA strand and thegrowing RNA strand that is base-paired to it have opposite5n3 directionality.By convention, the site at which RNA polymerase beginstranscription is numbered 1. Downstream denotes the direction in which a template DNA strand is transcribed (ormRNA translated); thus a downstream sequence is towardthe 3 end relative to the start site, considering the DNAstrand with the same polarity as the transcribed RNA.
Upstream denotes the opposite direction. Nucleotide positionsin the DNA sequence downstream from a start site are indicated by a positive () sign; those upstream, by a negative() sign.353 RNAstrand growthBaseOBaseO5HHOHOHH−OBasestrandOOBaseODNAtemplatePHHOHOHH−OBasePOOBaseOHHH3 HOHOHPolymerizationBaseOBaseOOHHOHHOHHPαO−OOPβO−OOPγO−O−Incoming rNTPBaseBase5▲ FIGURE 4-9 Polymerization of ribonucleotides by RNApolymerase during transcription. The ribonucleotide to beadded at the 3 end of a growing RNA strand is specified bybase pairing between the next base in the template DNA strandand the complementary incoming ribonucleoside triphosphate(rNTP).
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