tRNA & Ribosomes Copyright © 1999-2008 by Joyce J. Diwan. All rights reserved. Molecular Biochemistry II Molecular Biology Familiarity with basic concepts is assumed, including: nature of the genetic code maintenance of genes through DNA replication transcription of information from DNA to mRNA translation of mRNA into protein. DNA mRNA protein Purines & Pyrimidines N N N H N NH 2 HN N N H N O H 2 N N N H NH 2 O NH N H O O adenine (A) guanine (G) cytosine (C) uracil (U) N N N H N NH 2 H 3 C + HN N N H N O H 2 N CH 3 + N N H NH 2 O CH 3 + NH N H HN O O 1-methyladenine (m 1 A) 7-methylguanine (m 7 G) 3-methylcytosine (m 3 C) pseudouracil (Ψ) Nucleoside bases found in RNA: Nucleic acids are polymers of nucleotides. Each nucleotide includes a base that is either a purine (adenine or guanine), or a pyrimidine (cytosine, uracil, or thymine). Some nucleic acids contain modified bases. Examples: N N N H N NH 2 HN N N H N O H 2 N N N H NH 2 O NH N H O O adenine (A) guanine (G) cytosine (C) uracil (U) N N N H N NH 2 H 3 C + HN N N H N O H 2 N CH 3 + N N H NH 2 O CH 3 + NH N H HN O O 1-methyladenine (m 1 A) 7-methylguanine (m 7 G) 3-methylcytosine (m 3 C) pseudouracil (Ψ) Nucleoside bases found in RNA: Examples of modified bases found in tRNA: In a nucleotide, e.g., adenosine monophosphate (AMP), the base is bonded to a ribose sugar, which has a phosphate in ester linkage to the 5' hydroxyl. N N N N NH 2 adenine adenosine adenosine monophosphate (AMP) O OHOH HH H CH 2 H HO N N N H N NH 2 N N N N NH 2 O OHOH HH H CH 2 H OO 3 P − 2 ribose 5' adenine 4' 3' 2' 1' Nucleic acids have a backbone of alternating P i & ribose moieties. Phosphodiester linkages form as the 5' phosphate of one nucleotide forms an ester link with the 3' OH of the adjacent nucleotide. A short stretch of RNA is shown. N N N N NH 2 O OH O HH H CH 2 H ribose adenine P O O O O OHO HH H CH 2 H N N NH 2 O P O O O OP O O O − − − cytosine 5' 4' 3' 2' 1' ribose 3' 5' 3' end 5' end − (etc) nucleic acid Hydrogen bonds link 2 complementary nucleotide bases on separate nucleic acid strands, or on complementary portions of the same strand. Conventional base pairs: A & U (or T); C & G. In the diagram at left, H-bonds are in red. Bond lengths are inexact. The image at right is based on X-ray crystallography of tRNA Gln . H atoms are not shown. N N N H N O N N NH N O H H H H H guanine (G) cytosine (C) G C G − C base pair in tRNA Secondary structure Base pairing over extended stretches of complementary base sequences in two nucleic acid strands stabilizes secondary structure, such as the double helix of DNA. Stacking interactions between adjacent hydrophobic bases contribute to stabilization of such secondary structures. Each base interacts with its neighbors above and below, in the ladder-like arrangement of base pairs in the double helix, e.g., of DNA. Genetic code The genetic code is based on the sequence of bases along a nucleic acid. Each codon, a sequence of 3 bases in mRNA, codes for a particular amino acid, or for chain termination. Some amino acids are specified by 2 or more codons. Synonyms (multiple codons for the same amino acid) in most cases differ only in the 3 rd base. Similar codons tend to code for similar amino acids. Thus effects of mutation are minimized. Genetic Code 2 nd base1 st base U C A G 3 rd base UUU Phe UCU Ser UAU Tyr UGU Cys U UUC Phe UCC Ser UAC Tyr UGC Cys C UUA Leu UCA Ser UAA Stop UGA Stop A U UUG Leu UCG Ser UAG Stop UGG Trp G CUU Leu CCU Pro CAU His CGU Arg U CUC Leu CCC Pro CAC His CGC Arg C CUA Leu CCA Pro CAA Gln CGA Arg A C CUG Leu CCG Pro CAG Gln CGG Arg G AUU Ile ACU Thr AAU Asn AGU Ser U AUC Ile ACC Thr AAC Asn AGC Ser C AUA Ile ACA Thr AAA Lys AGA Arg A A AUG Met* ACG Thr AAG Lys AGG Arg G GUU Val GCU Ala GAU Asp GGU Gly U GUC Val GCC Ala GAC Asp GGC Gly C GUA Val GCA Ala GAA Glu GGA Gly A G GUG Val GCG Ala GAG Glu GGG Gly G *Met and initiation. [...]... NH2 H H OH H OH tRNA AMP 2 tRNA O H Aminoacyl-AMP In step 2, the 2' or 3' OH of the terminal adenosine of tRNA attacks the amino acid carbonyl C atom Adenine O O P O CH2 O− O H H Adenine H H OH 3’ 2’ O C O HC R NH3+ (terminal 3’nucleotide of appropriate tRNA) Aminoacyl -tRNA Aminoacyl -tRNA Synthetase - summary: 1 amino acid + ATP aminoacyl-AMP + PPi 2 aminoacyl-AMP + tRNA aminoacyl -tRNA + AMP The... &5 S RNAs 49 proteins Eukaryotic cytoplasmic ribosomes are larger and more complex than prokaryotic ribosomes Mitochondrial and chloroplast ribosomes differ from both examples shown 5S rRNA “crown” view displayed as ribbons & sticks PDB 1FFK Structures of large & small subunits of bacterial & eukaryotic ribosomes have been determined, by X-ray crystallography & by cryo-EM with image reconstruction Consistent... Aminoacyl -tRNA Synthetases arose early in evolution Early aaRSs probably recognized tRNAs only by their acceptor stems tRNA acceptor stem tRNA O O P O (terminal 3’nucleotide of appropriate tRNA) CH2 O− There are 2 families of Aminoacyl -tRNA Synthetases: Class I & Class II O H H O Adenine H H OH 3’ 2’ C O HC R NH3+ Aminoacyl -tRNA Two different ancestral proteins evolved into the 2 classes of aaRS enzymes,... different Aminoacyl -tRNA Synthetase (aaRS) for each amino acid Accurate translation of the genetic code depends on attachment of each amino acid to an appropriate tRNA Each aaRS recognizes its particular amino acid & tRNAs coding for that amino acid Identity elements: tRNA domains recognized by an aaRS Most identity elements are in the acceptor stem & anticodon loop anticodon loop Aminoacyl -tRNA Synthetases... the S atom in cysteine There is a selenocysteine tRNA that differs from other tRNAs, e.g., in having a slightly longer acceptor stem & a unique modified base in the anticodon loop tRNASec is loaded with serine via Seryl -tRNA Synthetase The serine moiety is then converted to selenocysteine by another enzyme, in a reaction involving selenophosphate Sec-tRNASec utilization during protein synthesis requires... #13 C Tertiary base Phe pairs in tRNA An example of non-standard H bond interactions that help to stabilize the L-shaped tertiary structure of a tRNA, in ball & stick & spacefill displays H atoms are not shown (From NDB file 1TN2) Some other RNAs, including viral RNAs & segments of ribosomal RNA, fold in pseudoknots, tertiary structures that mimic the 3D structure of tRNA Pseudoknots are similarly stabilized... control: Some Aminoacyl -tRNA Synthetases are known to have separate catalytic sites that release by hydrolysis inappropriate amino acids that are misacylated or mistransferred to tRNA E.g., the aa -tRNA Synthetase for isoleucine (IleRS) a small percentage of the time activates the closely related amino acid valine to valine-AMP After valine is transferred to tRNAIle, to form Val-tRNAIle, it is removed... not the larger Ile In some bacteria, editing of some misacylated tRNAs is carried out by separate proteins that may be evolutionary precursors to editing domains of aa -tRNA Synthetases Some amino acids are modified after being linked to a tRNA Examples: In prokaryotes the initiator tRNAfMet is first charged with methionine Methionyl -tRNA formyltransferase then catalyzes formylation of the methionine,... of the methionine, using tetrahydrofolate as formyl donor, to yield formylmethionyl-tRNAfMet In some prokaryotes, a non-discriminating aaRS loads aspartate onto tRNAAsn The aspartate moiety is then converted by an amidotransferase to asparagine, yielding Asn-tRNAAsn Glu-tRNAGln is similarly formed and converted to GlntRNAGln in such organisms Some proteins contain the unusual amino acid selenocysteine.. .tRNA The genetic code is read during translation via adapter molecules, tRNAs, that have 3-base anticodons complementary to codons in mRNA "Wobble" during reading of the mRNA allows some tRNAs to read multiple codons that differ only in the 3rd base There are 61 codons specifying 20 amino acids Minimally 31 tRNAs are required for translation, not counting the tRNA that codes for