Molecular Biology Fourth Edition Chapter 19 Ribosomes and Transfer RNA Lecture PowerPoint to accompany Robert F. Weaver Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Ribosomes E. coli ribosome is a two-part structure with a sedimentation coefficient of 70S Two subunits of this structure: –30S is the small subunit that decodes mRNA –50S subunit links amino acids together through peptide bonds

19-3 Fine Structure of 70S Ribosome T. thermophilus crystal structure of 70S ribosome in complex with mRNA analog and 3 tRNAs shows: –Positions and tertiary structures of all 3 rRNA and most proteins can be determined –Shapes and locations of tRNAs in A, P, and E sites are evident –Binding sites for tRNAs in ribosome are composed of rRNA, not protein –Anticodons of tRNAs in A and P sites approach each other closely enough to base-pair with adjacent codons bound to 30S subunit as mRNA kinks 45°

19-4 More Structural Detail Acceptor stems of tRNAs in A and P sites also approach each other closely (5 Å) in the peptidyl transferase pocket of 50S subunit Consistent with need for 2 stems to interact during peptide bond formation 12 contacts are seen between subunits, most RNA-RNA interactions

19-5 E. coli Ribosome Structure Crystal structure of E. coli ribosome contains 2 structures differing from each other by rigid body motions of ribosome domains relative to each other –Head of 30S particle rotates by 6 –12 rotation compared to T. thermophilus ribosome –Probably part of a ratchet action that occurs during translocation

19-6 Eukaryotic Ribosomes Eukaryotic cytoplasmic ribosomes are: –Larger –More complex Eukaryotic organellar ribosomes are smaller than prokaryotic ones

19-7 Ribosome Composition The E. coli 30 subunit contains –16S rRNA –21 proteins (S1 – S21) E. coli 50S subunit contains –5S rRNA –23S rRNA –34 proteins (L1 – L34) Eukaryotic cytoplasmic ribosomes are: –Larger –Contain more RNAs and proteins

19-8 Ribosome Assembly Assembly of the 30S ribosomal subunit in vitro begins with 16S rRNA Proteins join sequentially and cooperatively –Proteins added early in the process –Help later proteins to bind to the growing particle

19-9 Fine Structure of the 30S Subunit Sequence studies of 16S rRNA led to a proposal for secondary structure of the molecule X-ray crystallography studies have confirmed the conclusions of these studies –30S subunit with extensively base-paired 16S rRNA whose shape essentially outlines the whole particle –X-ray crystallography studies confirmed locations of most of the 30S ribosomal proteins

19-10 Interaction of the 30S Subunit with Antibiotics 30S ribosomal subunit plays 2 roles –Facilitates proper decoding between codons and aminoacyl-tRNA anticodons –Also participates in translocation Crystal structures of 30S subunits with 3 antibiotics interferring with these 2 roles shed light on translocation and decoding –Spectinomycin –Streptomycin –Paromomycin

19-11 Spectinomycin Spectinomycin binds to 30S subunit near the neck At this site, binding interferes with movement of the head Head movement is required for translocation

19-12 Streptomycin Streptomycin binds near the A site of 30S subunit Binding stabilizes the ram state of the ribosomes Fidelity of translation is reduced: –Allowing noncognate aminoacyl-tRNAs to bind easily to the A site –Preventing the shift to the restrictive state that is necessary for proofreading

19-13 Paromomycin Paromomycin binds in the minor groove of 16S rRNA H44 helix near the A site This binding flips out bases A1492 and A1493 to stabilize base pairing between codon and anti- codon –Flipping out process normally requires energy –Paromomycin forces it to occur and keeps the stabilizing bases in place State of the A site stabilizes codon-anticodon interaction, including interaction between noncognate codons and anticodons with decline in fidelity

19-14 Interaction of the 30S Subunit with Initiation Factors X-ray crystal structure of IF1 bound to the 30S ribosomal subunit shows IF1 binds to the A site In that position IF1: –Blocks fMet-tRNA from binding to the A site –May also actively promote fMet-tRNA binding to P site through interaction between IF1 and IF2 IF1 also interacts closely with helix H44 of the 30S subunit IF accelerates both association and dissociation of the ribosomal subunits

19-15 Fine Structure of the 50S Subunit Crystal structure of the 50S ribosomal subunit has been determined to 2.4 Å Structure reveals relatively few proteins at interface between ribosomal subunits –No proteins within 18 Å of peptidyl transferase active center tagged with a transition state analog –2’-OH group of tRNA in the P site is very well positioned to form a hydrogen bond to amino group of aminoacyl-tRNA in A site

‘-Hydroxyl Group Role 2’-OH group of tRNA in the P site –Forms a hydrogen bond to amino group of aminoacyl- tRNA in A site –Helps catalyze peptidyl transferase reaction Removal of this hydroxyl group eliminates peptidyl transferase activity Removal of the 2’-OH of A2451 of the 23S rRNA inhibits peptidyl transferase activity May also participate in catalysis by: –Hydrogen bonding –Helping to position reactants properly for catalysis

S Exit Tunnel Exit tunnel through the 50S subunit –Just wide enough to allow a protein  -helix to pass –Walls of tunnel are made of RNA –Hydrophobicity is likely to allow exposed hydrophobic side chains of nascent polypeptide to slide through easily

19-18 Polysomes Most mRNAs are translated by more than one ribosome at at time A structure in which many ribosomes translate mRNA in tandem is called a polysome Eukaryotic polysomes are found in the cytoplasm In prokaryotes, transcription of a gene and translation of the resulting mRNA occur simultaneously Many polysomes are found associated with an active gene

Transfer RNA An adaptor molecule was proposed that could serve as a mediator between string of nucleotides in DNA or RNA and the string of amino acids in the corresponding protein The adaptor contained 2 or 3 nucleotides that could pair with nucleotides in codons

19-20 The Discovery of tRNA Transfer RNA was discovered as a small species independent of ribosomes This small species could be charged with an amino acid That species could then pass the amino acid to a growing polypeptide

19-21 tRNA Structure All tRNAs share a common secondary structure represented by a cloverleaf Four base-paired stems define three stem- loops –D loop –Anticodon loop –T loop The acceptor stem is the site to which amino acids are added in the charging step

19-22 tRNA Shape tRNAs share a common three-dimensional shape resembling an inverted L This shape maximizes stability by lining up the base pairs: –In the D stem with those in the anticodon stem –In the T stem with those in the acceptor stem Anticodon of the tRNA protrudes from the side of the anticodon loop –Anticodon is twisted into a shape that base- pairs with corresponding codon in mRNA

19-23 Modified Nucleosides in tRNA

19-24 Recognition of tRNA Acceptor Stem Biochemical and genetic experiments have demonstrated the importance of the acceptor stem in recognition of a tRNA by its cognate aminoacyl-tRNA synthetase Changing one base pair in the acceptor stem can change the charging specificity

19-25 The Anticodon Biochemical and genetic experiments have shown that anticodon, like acceptor stem, is an important element in charging specificity Sometimes the anticodon can be the absolute determinant of specificity

19-26 Structures of Synthetase-tRNA Complexes Crystallography has shown that synthetase- tRNA interactions differ between the 2 classes of aminoacyl-tRNA synthetases –Class I synthetases Pockets for acceptor stem and anticodon of their cognate tRNA Approach the tRNAs from the D loop and acceptor stem minor groove side –Class II synthetases Also have pockets for acceptor stem and anticodon Approach tRNA from opposite including the variable arm and the major groove of the acceptor stem

19-27 Proofreading and Editing Amino acid selectivity of at least some aminoacyl- tRNA synthetases is controlled by a double-sieve mechanism –1 st sieve is coarse excluding amino acids too big Enzyme accomplishes this with an active site for activation of amino acids just big enough to accommodate the cognate amino acid, not larger amino acids –2 nd sieve is fine, degrades too small aminoacyl-AMPs Done with a second active site, the editing site, admits small aminoacyl-AMPs and hydrolyzes them Cognate aminoacyl-AMP is too big to fit into the editing site Enzyme transfers the activated amino acid to its cognate tRNA