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tRNA & Ribosomes Molecular Biochemistry II

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1 tRNA & Ribosomes Molecular Biochemistry II
Copyright © by Joyce J. Diwan. All rights reserved.

2 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.

3 Purines & Pyrimidines 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).

4 Some nucleic acids contain modified bases. Examples:

5 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.

6 Nucleic acids have a backbone of alternating Pi & 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.

7 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 tRNAGln. H atoms are not shown.

8 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.

9 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 3rd base. Similar codons tend to code for similar amino acids. Thus effects of mutation are minimized.


11 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 chain initiation. Mammalian cells produce more than 150 tRNAs.

12 RNA structure: Most RNA molecules have secondary structure, consisting of stem & loop domains. Double helical stem domains arise from base pairing between complementary stretches of bases within the same strand. These stem structures are stabilized by stacking interactions as well as base pairing, as in DNA. Loop domains occur where lack of complementarity or the presence of modified bases prevents base pairing.

13 The “cloverleaf” model of tRNA emphasizes the two major types of secondary structure, stems & loops.
tRNAs typically include many modified bases, particularly in loop domains.

14 RNA tertiary structure depends on interactions of bases at distant sites.
These interactions generally involve non-standard base pairing and/or interactions involving three or more bases. Unpaired adenosines (not involved in conventional base pairing) predominate in participating in non- standard interactions that stabilize tertiary RNA structures. tRNAs have an L-shaped tertiary structure.

15 Extending from the acceptor stem, the 3' end of each tRNA has the sequence CCA.
The appropriate amino acid is attached to the ribose of the terminal adenosine (A, in red) at the 3' end. The anticodon loop is at the opposite end of the L shape.

16 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).

17 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 by non-standard H-bond interactions. Explore tRNAPhe with Chime (PDB file 1TRA).

18 Aminoacyl-tRNA Synthetases catalyze linkage of the appropriate amino acid to each tRNA.
The reaction occurs in 2 steps. In step 1, an O atom of the amino acid a-carboxyl attacks the P atom of the initial phosphate of ATP.

19 In step 2, the 2' or 3' OH of the terminal adenosine of tRNA attacks the amino acid carbonyl C atom.

20 Aminoacyl-tRNA Synthetase - summary:
1. amino acid + ATP  aminoacyl-AMP + PPi 2. aminoacyl-AMP + tRNA  aminoacyl-tRNA + AMP The 2-step reaction is spontaneous overall, because the concentration of PPi is kept low by its hydrolysis, catalyzed by Pyrophosphatase.

21 There is generally a 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. Aminoacyl-tRNA Synthetases arose early in evolution. Early aaRSs probably recognized tRNAs only by their acceptor stems.

22 There are 2 families of Aminoacyl-tRNA Synthetases: Class I & Class II.
Two different ancestral proteins evolved into the 2 classes of aaRS enzymes, which differ in the architecture of their active site domains. They bind to opposite sides of the tRNA acceptor stem, aminoacylating a different OH of the tRNA (2' or 3').

23 Class I aaRSs: Identity elements usually include residues of the anticodon loop & acceptor stem. Class I aaRSs aminoacylate the 2'-OH of adenosine at their 3' end. Class II aaRSs: Identity elements for some Class II enzymes do not include the anticodon domain. Class II aaRSs tend to aminoacylate the 3'-OH of adenosine at their 3' end.

24 Proofreading/quality control:
Some Aminoacyl-tRNA Synthetases are known to have separate catalytic sites that release by hydrolysis inappropriate amino acids that are misacylated or mis-transferred 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 by hydrolysis at a separate active site of IleRS that accommodates Val but 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.

25 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, 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 amido-transferase to asparagine, yielding Asn-tRNAAsn. Glu-tRNAGln is similarly formed and converted to Gln-tRNAGln in such organisms.

26 Some proteins contain the unusual amino acid selenocysteine (Sec), with selenium substituting for 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 special elongation factors because the codon for selenocysteine is UGA, which normally is a stop codon.

27 Other roles of aminoacyl-tRNA synthetases:
In some organisms, Aminoacyl-tRNA Synthetases (aaRSs) have evolved to take on signaling roles in addition to the catalytic role of joining an amino acid to the correct tRNA. Examples have been identified of particular aaRSs that regulate transcription, translation or intron splicing through binding to DNA or RNA.

28 Proteolytic cleavage of the human aaRSTyr yields a cytokine that stimulates angiogenesis.
A truncated form of the human aaRSTrp inhibits angiogenesis. Regulation of apoptosis by the human aaRSGln is dependent on the concentration of its substrate glutamine. Several mammalian Aminoacyl-tRNA Synthetases associate with other proteins to form large macromolecular complexes whose roles are actively being investigated.

29 Ribosome Composition (S = sedimentation coefficient)
Eukaryotic cytoplasmic ribosomes are larger and more complex than prokaryotic ribosomes. Mitochondrial and chloroplast ribosomes differ from both examples shown.

30 Structures of large & small subunits of bacterial & eukaryotic ribosomes have been determined, by X-ray crystallography & by cryo-EM with image reconstruction. Consistent with predicted base pairing, X-ray crystal structures indicate that ribosomal RNAs (rRNAs) have extensive secondary structure.

31 Structure of the E. coli Ribosome
The cutaway view at right shows positions of tRNA (P, E sites) & mRNA (as orange beads). EF-G will be discussed later. This figure was provided by Joachim Frank, whose lab at the Wadsworth Center carried out the cryo-EM and 3D image reconstruction on which the images are based.

32 Small Ribosomal Subunit
In the translation complex, mRNA threads through a tunnel in the small ribosomal subunit. tRNA binding sites are in a cleft in the small subunit. The 3' end of the 16S rRNA of the bacterial small subunit is involved in mRNA binding. The small ribosomal subunit is relatively flexible, assuming different conformations. E.g., the 30S subunit of a bacterial ribosome was found to undergo specific conformational changes when interacting with a translation initiation factor.

33 Small ribosomal subunit of a thermophilic bacterium:
rRNA in monochrome; proteins in varied colors. spacefill display ribbons The overall shape of the 30S ribosomal subunit is largely determined by the rRNA. The rRNA mainly consists of double helices (stems) connected by single-stranded loops. The proteins generally have globular domains, as well as long extensions that interact with rRNA and may stabilize interactions between RNA helices.

34 Large ribosome subunit:
The interior of the large subunit is mostly RNA. Proteins are distributed mainly on the surface. Some proteins have long tails that extend into the interior of the complex. These tails, which are highly basic, interact with the negatively charged RNA.

35 The active site domain for peptide bond formation is essentially devoid of protein.
The peptidyl transferase function is attributed to 23S rRNA, making this RNA a "ribozyme."

36 Protein synthesis takes place in a cavity within the ribosome, between small & large subunits.
Nascent polypeptides emerge through a tunnel in the large subunit. The tunnel lumen is lined with rRNA helices and some ribosomal proteins.

37 Catalysis of protein synthesis and movement of the ribosome relative to messenger RNA are accompanied by changes in ribosome conformation. EM & X-ray crystallographic studies, carried out in the presence & absence of initiation & elongation factors as well as inhibitors of protein synthesis, have revealed conformational changes in rRNA. Thus rRNA participates in conformational coupling in addition to its structural & catalytic roles. tRNAs also undergo substantial conformational changes within ribosomal binding sites during protein synthesis. Explore the large ribosomal subunit.

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