Ribosome sequence determination

Because the sequencing of DNA has become so straightforward, genes of several ribosomal proteins have been sequenced to allow an independent determination of the primary sequence of some of the proteins (Post et ai, 1979 Olins and Nomura, 1981). These studies have confirmed the sequences previously elucidated by protein chemical techniques. In the case of SI (the largest of all E. coli proteins), the combination of amino acid- and nucleotide-sequence determinations was used to provide the sequence (Schnier et ai, 1982). 

Biswas, R Jiang, X. Pacchia, A. L. Dougherty, J. R Peltz, S. W. The human immunodeficiency virus type 1 ribosomal frameshifting site is an invariant sequence determinant and an important target for antiviral therapy. J. Virol. 2004, 78, 2082-2087. 

Transfer RNA, tRNA, soluble RNA, sRNA. Low mol wt 23,000-27,000 approx 75-85 nucleotides. Each tRNA is specific for and binds with a particular amino acid more than one may exist for each amino acid. Performs three functions during protein synthesis binds with its specific amino acid recognizes the corresponding codon on mRNA and places the amino acid in the correct position for attach -ment to the polypeptide chain being formed binds the poly -peptide to the ribosome. First determination of total structure of a transfer RNA (yeast alanine tRNA) Holley et aL. Science 147, 1462 (1965). Reviews of structure and function Miura, Specificity in the Structure of Transfer RNA in fVogr, Nucleic Acid Res. Mol. BioL 6, 39-82 (1967) Cramer, Three-Dimensional Structure of tRNA , ibid. 11, 391-421 (1971) Nucleic Acid Sequence Analysis, S. Mandeles (Columbia University Press, New York, 1972) pp 256-280 Nishi-mura, "Transfer RNA Structure and Biosynthesis in MTP Int. Rev. Sci Biochem.. Ser. One vol. 6, K. Burton, Ed. (University Park Press, Baltimore, 1974) pp 289-322 A. Rich, V. L. Raj Bhandary, Ann. Rev. Biochem. 45, 805-860 (1976) P. F. Agris, The Modified Nucleosides of Transfer RNA, IT (A. R. Liss, New York, 1983) 220 pp. 

Nucleic acids can play roles farbeyond merely harbouring the coding information for proteins. Single-stranded nucleic acids can fold into intricate structures capable of molecular recognition and even catalysis. Three-dimensional structures are specified by the primary structure, namely the deoxynucleotide (or nucleotide, for RNA) sequence (5 - 3, by analogy to the situation in which the amino-acid residue sequence determines the three-dimensional structures of polypeptides. In nature, transfer RNAs (tRNAs) use their three-dimensional shape for molecular recognition, while some ribosomal RNAs (rRNAs) are able to catalyse crucial steps even within the protein synthetic pathways themselves. 

Translation. Translation is the process by which the instructions encoded in mRNA are used, in conjunction with tRNA and ribosomes, to determine the sequence of amino acids during protein synthesis... 

Transfer RNA (tRNA) carries amino acids in an activated form to the ribosome for peptide bond formation, in a sequence determined by the mRNA template. There is at least one fRNA for each of the 20 amino acids. Transfer RNA molecules are relatively small as nucleic acids go, with about 70 to 90 nucleotide units. Each fRNA has a three-base sequence, C—C—A, at the 3 hydroxyl end, where the amino acid is attached as an ester. Each fRNA also has an anticodon loop quite remote from the amino acid attachment site. This loop contains seven nucleotides, the middle three of which are complementary to the three-base code word on the mRNA for that particular amino acid. 

As mentioned above, it was concluded from immunological studies that there is no extensive homology among E. coli ribosomal proteins, and this has also been confirmed by the amino acid sequence analyses which have so far been performed. With the exception of the two pairs of proteins described above, only very short regions of homology (up to five residues) occur. This finding is based on the sequence determination of about 3000 amino acid residues, i.e. 35 % of the approximately 8000 amino acids present in the E. coli ribosome. 

Among the achievements that have been made during the last few years are the complete sequence determination of all the ribosomal components from Escherichia coli the determination of the shape of the ribosomal particles and the location of many ribosomal components and the gradual understanding of the secondary structures of the ribosomal RNA-molecules. Many areas nevertheless remain poorly understood the quaternary structure of the whole ribosome the tertiary structure of its components and the cooperative interactions of these components during protein biosynthesis. 

In contrast, RNA occurs in multiple copies and various forms (Table 11.2). Cells contain up to eight times as much RNA as DNA. RNA has a number of important biological functions, and on this basis, RNA molecules are categorized into several major types messenger RNA, ribosomal RNA, and transfer RNA. Eukaryotic cells contain an additional type, small nuclear RNA (snRNA). With these basic definitions in mind, let s now briefly consider the chemical and structural nature of DNA and the various RNAs. Chapter 12 elaborates on methods to determine the primary structure of nucleic acids by sequencing methods and discusses the secondary and tertiary structures of DNA and RNA. Part rV, Information Transfer, includes a detailed treatment of the dynamic role of nucleic acids in the molecular biology of the cell. 

Elongation is a cycUc process on the ribosome in which one amino acid at a time is added to the nascent peptide chain. The peptide sequence is determined by the order of the codons in the mRNA. Elongation involves several steps catalyzed by proteins called elongation factors (EFs). These steps are (1) binding of aminoacyl-tRNA to the A site, (2) peptide bond formation, and (3) translocation. 

The only other E. coli ribosomal protein whose crystallization has so far been reported is L29 (Appelt et al., 1981). On the other hand, attempts to crystallize ribosomal proteins from the thermophilic Bacillus stearothermophilus have been more successful. Protein BL17, which according to its amino acid sequence (Kimura et al., 1980) corresponds to protein L9 from the E. coli ribosome (Kimura et al., 1982), was the first intact ribosomal protein to give crystals useful for X-ray structural analysis (Appelt et al., 1979). Several other B. stearothermophUus ribosomal proteins, namely BL6 and BL30 (Appelt eteU., 1981,1983) from the large and BS5 (Appelt et al., 1983) from the small subunit have been crystallized, and the determination of their three-dimensional structure at a resolution of better than 3 A is now in progress. Furthermore, crystals of aB. stearothermophilus ribosomal protein complex, which corresponds to the complex (L7/L12)4 LIO from E. coli ribosome, have been obtained (Liljas and Newcomer, 1981). 

Secretory pathway. Ribosomes that synthesize a protein with a signal peptide for the ER settle on the ER (see p. 228). The peptide chain is transferred into the lumen of the rER. The presence or absence of other signal sequences and signal regions determines the subsequent transport pathway. 

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