Lodish H. - Molecular Cell Biology (5ed, Freeman, 2003) (794361), страница 53
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The base in the third (or wobble)position of an mRNA codon often forms a nonstandard basepair with the base in the first (or wobble) position of a tRNAanticodon. Wobble pairing allows a tRNA to recognize more thanone mRNA codon (top); conversely, it allows a codon to berecognized by more than one kind of tRNA (bottom), althougheach tRNA will bear the same amino acid. Note that a tRNAwith I (inosine) in the wobble position can “read” (becomepaired with) three different codons, and a tRNA with G or U inthe wobble position can read two codons. Although A istheoretically possible in the wobble position of the anticodon,it is almost never found in nature.123(I), a deaminated product of adenine, at this position. Inosine can form nonstandard base pairs with A, C, and U. AtRNA with inosine in the wobble position thus can recognizethe corresponding mRNA codons with A, C, or U in the third(wobble) position (see Figure 4-23).
For this reason, inosinecontaining tRNAs are heavily employed in translation of thesynonymous codons that specify a single amino acid. For example, four of the six codons for leucine (CUA, CUC, CUU,and UUA) are all recognized by the same tRNA with the anticodon 3-GAI-5; the inosine in the wobble position formsnonstandard base pairs with the third base in the four codons.In the case of the UUA codon, a nonstandard G·U pair alsoforms between position 3 of the anticodon and position 1 ofthe codon.Aminoacyl-tRNA Synthetases Activate AminoAcids by Covalently Linking Them to tRNAsRecognition of the codon or codons specifying a given aminoacid by a particular tRNA is actually the second step in decoding the genetic message. The first step, attachment of theappropriate amino acid to a tRNA, is catalyzed by a specificaminoacyl-tRNA synthetase. Each of the 20 different synthetases recognizes one amino acid and all its compatible, orcognate, tRNAs.
These coupling enzymes link an amino acidto the free 2 or 3 hydroxyl of the adenosine at the 3 terminus of tRNA molecules by an ATP-requiring reaction. Inthis reaction, the amino acid is linked to the tRNA by a highenergy bond and thus is said to be activated. The energy ofthis bond subsequently drives formation of the peptide bondslinking adjacent amino acids in a growing polypeptide chain.The equilibrium of the aminoacylation reaction is driven further toward activation of the amino acid by hydrolysis of thehigh-energy phosphoanhydride bond in the released pyrophosphate (see Figure 4-21).Because some amino acids are so similar structurally,aminoacyl-tRNA synthetases sometimes make mistakes.These are corrected, however, by the enzymes themselves,which have a proofreading activity that checks the fit in theiramino acid–binding pocket.
If the wrong amino acid becomes attached to a tRNA, the bound synthetase catalyzesremoval of the amino acid from the tRNA. This crucial function helps guarantee that a tRNA delivers the correct aminoacid to the protein-synthesizing machinery. The overall errorrate for translation in E. coli is very low, approximately 1 per50,000 codons, evidence of the importance of proofreadingby aminoacyl-tRNA synthetases.Ribosomes Are Protein-Synthesizing MachinesIf the many components that participate in translatingmRNA had to interact in free solution, the likelihood of simultaneous collisions occurring would be so low that therate of amino acid polymerization would be very slow.
Theefficiency of translation is greatly increased by the binding ofthe mRNA and the individual aminoacyl-tRNAs to the most124CHAPTER 4 • Basic Molecular Genetic Mechanismsabundant RNA-protein complex in the cell, the ribosome,which directs elongation of a polypeptide at a rate of three tofive amino acids added per second. Small proteins of100–200 amino acids are therefore made in a minute or less.On the other hand, it takes 2–3 hours to make the largestknown protein, titin, which is found in muscle and containsabout 30,000 amino acid residues. The cellular machine thataccomplishes this task must be precise and persistent.With the aid of the electron microscope, ribosomes werefirst discovered as small, discrete, RNA-rich particles in cellsthat secrete large amounts of protein.
However, their role inprotein synthesis was not recognized until reasonably pureribosome preparations were obtained. In vitro radiolabelingexperiments with such preparations showed that radioactiveamino acids first were incorporated into growing polypeptide chains that were associated with ribosomes before appearing in finished chains.A ribosome is composed of three (in bacteria) or four (ineukaryotes) different rRNA molecules and as many as 83proteins, organized into a large subunit and a small subunit(Figure 4-24).
The ribosomal subunits and the rRNA molecules are commonly designated in Svedberg units (S), ameasure of the sedimentation rate of suspended particles cen-ProteinsrRNAtrifuged under standard conditions. The small ribosomalsubunit contains a single rRNA molecule, referred to as smallrRNA. The large subunit contains a molecule of large rRNAand one molecule of 5S rRNA, plus an additional moleculeof 5.8S rRNA in vertebrates. The lengths of the rRNA molecules, the quantity of proteins in each subunit, and consequently the sizes of the subunits differ in bacterial andeukaryotic cells. The assembled ribosome is 70S in bacteriaand 80S in vertebrates. But more interesting than these differences are the great structural and functional similaritiesbetween ribosomes from all species.
This consistency is another reflection of the common evolutionary origin of themost basic constituents of living cells.The sequences of the small and large rRNAs from severalthousand organisms are now known. Although the primarynucleotide sequences of these rRNAs vary considerably, thesame parts of each type of rRNA theoretically can form basepaired stem-loops, which would generate a similar threedimensional structure for each rRNA in all organisms. Theactual three-dimensional structures of bacterial rRNAs fromThermus thermopolis recently have been determined by xray crystallography of the 70S ribosome.
The multiple, muchsmaller ribosomal proteins for the most part are associatedSubunitsAssembledribosomes5S23SProkaryotic+23S(2900 rNTs)Total: 315S(120 rNTs)50S+Total: 2116S16S(1500 rNTs)70S30SEukaryotic (vertebrate)5S28S+5.8STotal: 5028S5S28S : 5.8S(4800 rNTs, 160 rNTs) (120 rNTs)5.8S60S+Total: 3318S(1900 rNTs)▲ FIGURE 4-24 The general structure of ribosomes inprokaryotes and eukaryotes.
In all cells, each ribosomeconsists of a large and a small subunit. The two subunitscontain rRNAs (red) of different lengths, as well as a different setof proteins. All ribosomes contain two major rRNA molecules18S40S80S(23S and 16S rRNA in bacteria; 28S and 18S rRNA in vertebrates)and a 5S rRNA. The large subunit of vertebrate ribosomes alsocontains a 5.8S rRNA base-paired to the 28S rRNA. The numberof ribonucleotides (rNTs) in each rRNA type is indicated.4.5 • Stepwise Synthesis of Proteins on Ribosomeswith the surface of the rRNAs.
Although the number of protein molecules in ribosomes greatly exceeds the number ofRNA molecules, RNA constitutes about 60 percent of themass of a ribosome.During translation, a ribosome moves along an mRNAchain, interacting with various protein factors and tRNAsand very likely undergoing large conformational changes.Despite the complexity of the ribosome, great progress hasbeen made in determining the overall structure of bacterialribosomes and in identifying various reactive sites. X-raycrystallographic studies on the T.
thermophilus 70S ribosome, for instance, not only have revealed the dimensionsand overall shape of the ribosomal subunits but also have localized the positions of tRNAs bound to the ribosome duringelongation of a growing protein chain. In addition, powerful chemical techniques such as footprinting, which is described in Chapter 11, have been used to identify specificnucleotide sequences in rRNAs that bind to protein or another RNA. Some 40 years after the initial discovery of ribosomes, their overall structure and functioning duringprotein synthesis are finally becoming clear, as we describe inthe next section.KEY CONCEPTS OF SECTION 4.4The Three Roles of RNA in TranslationGenetic information is transcribed from DNA intomRNA in the form of a comma-less, overlapping, degenerate triplet code.■Each amino acid is encoded by one or more threenucleotide sequences (codons) in mRNA.
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