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

19.1 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 (Chapter. 17) 50S subunit links amino acids together through peptide bonds (Chapter 18)

Eukaryotic Ribosomes Eukaryotic cytoplasmic ribosomes are: Larger More complex Eukaryotic organellar ribosomes are smaller than prokaryotic ones

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

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

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 (codon-anticodon interaction) Spectinomycin- inhibit translocation Streptomycin- cause errors in translation Paromomycin – increase error rate by another mechanism

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

Streptomycin Streptomycin binds near the A site of 30S subunit Binding stabilizes the ram (ribosome ambiguity) 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

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

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

Fig. 19.21

19.2 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

The Discovery of tRNA Transfer RNA was discovered as a small species independent of ribosomes Zamecnik and et al.,1957 pH5 enzyme fraction works with ribosomes to direct translation of added mRNAs. This small species could be charged with an amino acid That species could then pass the amino acid to a growing polypeptide

Charging of tRNA with an amino acid Mix RNA with pH5 enzyme, ATP and [14C]Leucine Charging of tRNA with an amino acid

Mix [14C]Leucine-charged pH 5 RNA with microsome Incorporation of leucine from leucyl-tRNA in to the nascent protein on ribosome

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

Fig. 19.24

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

Modified Nucleosides in tRNA

Are tRNAs made with modified bases, or are the bases modified after transcription? tRNA are made in the same way that other RNAs are made, with the four standard bases. Multiple enzymes modify the bases

What effects, if any, do these modifications have on tRNA function? At least two tRNAs have been made in vitro with four normal, unmodified bases, and they were unable to bind amino acids Each modification may have subtle effects on the efficiency of charging and tRNA usage

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

AMP/amino acid coupling AMP/tRNA displacement

Ribosome recognize the tRNA but not the amino acid Lipmann, Benzer, von Ehrenstein and et al, 1962 Cysteinyl-tRNAcys →Alanyl-tRNAcys In vitro translation using poly(UGU) as template—this does not contain any codon for alanine Codon for ala is GCN, cysteine is UGU Alanine is incorporated instead of cysteine Fig. 19.37

It is the nature of the tRNA that matters Fig. 19.28 It is the nature of the tRNA that matters

The acceptor stem ? The anticodon ? Given that the secondary and tertiary structures of all tRNA are essentially the same, what base sequences in tRNA do the synthetases recognize when they are selecting one tRNA out of a pool of over 20? The acceptor stem ? The anticodon ?

The acceptor stem 1973, Smith and Celis found: GlnRS and TyrRS binding to tRNA differ only in one base 73 from G to A in suppressor tRNA assay

The acceptor stem 1988, Hou and Schimmel found: tRNAAla’s anticodn mutated to 5’-CUA-3’, so it inserts alanine in response to the amber codon UAG. Insert amber codon in codon 10 of trpA gene The mutation could be suppressed only by a tRNA that could insert an alanine in response to amber codon Any other amino acid in position 10 yielded an inactive protein G3-U70 base pair is a key determinant of charging synthetases

3

Further evidence tRNAcys/CUA and tRNAphe/CUA (CUA is anti-codon) Has C3-G70 base pair in their acceptor stem Change C3-G70 to G3-U70, then convert the tRNA to tRNAAla/CUA

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

The first base in the anticodon (the wobble position) was the Tab. 19.1 One different base The first base in the anticodon (the wobble position) was the most sensitive and had drastic effect on charging

Recognition of tRNAs by aminoacyl-tRNA synthetase: The second genetic code

1958, Pauling used thermodynamics and found: Ile and val only differ in –CH2 group and one-fifth Val-tRNAile would be made In fact, 1/150 amino acid is activated by IleRS to make Val and 1/3,000 aminoacyl-tRNA is Val-tRNAile

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

Proofreading and Editing Amino acid selectivity of at least some aminoacyl-tRNA synthetases is controlled by a double-sieve mechanism 1st 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 2nd 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

Fig. 19.31

The space between Trp232 and Tyr386 is just big enough to Fig. 19.33 The space between Trp232 and Tyr386 is just big enough to accommodate valine but not Ile (too big) Abolish this region could abolish editing but not activation activity