© 2014 Pearson Education, Inc. Chapter 15 Opener Translation

© 2014 Pearson Education, Inc. Translation Translation is mostly energy cost in cell, up to 80% in rapidly growing bacteria The translation machinery is composed of 4 primary components: mRNAs, tRNAs, aminoacyl-tRNA synthetases and ribosome. Polypeptide chains are specified by open reading frames (ORF).

© 2014 Pearson Education, Inc. Figure 15-1 Three possible reading frames of the E. coli trp reader sequence. Start codon in prokaryote 5’ AUG 3’ (mostly) 5’ GUG 3’ 5’ UUG 3’

© 2014 Pearson Education, Inc. Figure 15-2a Structure of mRNA. A polycistronic prokaryotic message with three ORFs. RBS (ribosome binding site): Shine-Dalgarno sequence (5’AGGAGG 3’) – recruit ribosome

© 2014 Pearson Education, Inc. Figure 15-2b Structure of mRNA A monocistronic eukaryotic message with 5’ Cap and 3’ poly A tail. 5’ cap recruits ribosome This sequence stimulates translation.

© 2014 Pearson Education, Inc. Figure 15-3 This modified bases improve tRNA function.

© 2014 Pearson Education, Inc. Figure 15-4 tRNAs are adapters between codons and amino acids (Pseudouridine loop) (Dihydrouridine loop)

© 2014 Pearson Education, Inc. Figure 15-5 Clover shape L shape Actual 3D structure (ribbon representation)

© 2014 Pearson Education, Inc. Figure 15-5c

© 2014 Pearson Education, Inc. Figure 15-6a

© 2014 Pearson Education, Inc. Figure 15-6b

© 2014 Pearson Education, Inc. Table 15-1

© 2014 Pearson Education, Inc. Figure 15-7

© 2014 Pearson Education, Inc. Figure 15-8

© 2014 Pearson Education, Inc. Figure 15-9 mistake tRNA charging is less than 1 in 1000tRNA Although two amino acids are similar in shape and size, OH of tyrosine is distinguished by tyrosyl- tRNA synthetase.

© 2014 Pearson Education, Inc. Figure 15-10

© 2014 Pearson Education, Inc. Box

© 2014 Pearson Education, Inc. Figure 15-11

© 2014 Pearson Education, Inc. Figure 15-12

© 2014 Pearson Education, Inc. Figure 15-13

© 2014 Pearson Education, Inc. Figure Overview of the events of translation: ribosome cycle

© 2014 Pearson Education, Inc. Figure Polyribosome

© 2014 Pearson Education, Inc. Figure 15-16

© 2014 Pearson Education, Inc. Figure S and 30S of prokaryote ribosome

© 2014 Pearson Education, Inc. Figure Ribosome has three tRNA binding sites

© 2014 Pearson Education, Inc. Figure tRNA in E P A sites Peptidyl transferase

© 2014 Pearson Education, Inc. Figure 15-19a

© 2014 Pearson Education, Inc. Figure 15-19b

© 2014 Pearson Education, Inc. Figure 15-19c Peptidyl transferase

© 2014 Pearson Education, Inc. Figure 15-19d Decoding center (anticodon) in 30S of ribosome tRNA

© 2014 Pearson Education, Inc. Figure Structure of mRNA and tRNA mRNA

© 2014 Pearson Education, Inc. Figure 15-21

© 2014 Pearson Education, Inc. Figure 15-22

© 2014 Pearson Education, Inc. Figure 15-23

© 2014 Pearson Education, Inc. Figure 15-24

© 2014 Pearson Education, Inc. Figure 15-25

© 2014 Pearson Education, Inc. In prokaryotes (GTPase: IF2) IF1 prevents tRNAs from binding to portion of small subunit that will be part of A site.

© 2014 Pearson Education, Inc.

Figure In eukaryotes Assembly of small ribosomal subunit and Initiator tRNA onto the mRNA 48S PIC(preinitiation complex)

© 2014 Pearson Education, Inc. Figure Circularization is mediated by interactions between eIF4G, the poly-A-binding protein and poly-A-tail.

© 2014 Pearson Education, Inc. Figure 15-28

© 2014 Pearson Education, Inc. Identification of initiating AUG by the 48S PIC(preinitiation complex) and large subunit joining eukaryotic translation initiation ATP dependent process of scan down stimulated by eIF4A/B- associated RNA helicase In eukaryotes 48S PIC(preinitiation complex)

© 2014 Pearson Education, Inc.

Figure Summary of the steps of translation elongation (in prokaryote, studied a lot and very similar also in eukaryotes)

© 2014 Pearson Education, Inc. Figure 15-30

© 2014 Pearson Education, Inc. Figure 15-31

© 2014 Pearson Education, Inc. Figure 15-31a Three mechanisms to ensure correct pairing between the tRNA and the mRNA Additional hydrogen bonds are formed between two adenine residues of the 16S rRNA and the minor groove of anticodon

© 2014 Pearson Education, Inc. Correct base pairing facilitates EF-Tu factors to interact with factor binding center to inducing GTP hydrolysis

© 2014 Pearson Education, Inc.

Figure 15-31c Only correctly base paired aminoacyl-tRNAs remain associated with ribosome as they rotate into correct position for peptide bond formation. This rotation is referred to as tRNA accommodation. rotation

© 2014 Pearson Education, Inc. Figure RNA surrounds the peptidyl transferase center of the large ribosomal subunit

© 2014 Pearson Education, Inc. Figure Proposed for the 2’ OH of the P-site tRNA in peptide bond formation

© 2014 Pearson Education, Inc. Figure Peptide bond formation (by peptidyl transferase) initiates translocation in the large subunit. EF-G stimulation of translocation requires GTP hydrolysis

© 2014 Pearson Education, Inc. Figure 15-34a

© 2014 Pearson Education, Inc. Figure 15-34b

© 2014 Pearson Education, Inc. Figure 15-34c

© 2014 Pearson Education, Inc. Figure 15-34d

© 2014 Pearson Education, Inc. Figure 15-34e

© 2014 Pearson Education, Inc. Figure EF-Tu-GDPNP(analog of GDP)- Phe-tRNA EF-Tu-GDP

© 2014 Pearson Education, Inc. Figure EF-Ts stimulates release of GDP from EF-Tu. EF-Tu-EF-Ts EF-Ts EF-Tu-GTP EF-Tu-GTP bound aminoacyl tRNA

© 2014 Pearson Education, Inc. Figure Release factors terminate translation in response to stop codons Two classes of releasing factor Class I recognize stop codon and trigger hydrolysis of peptide chain from tRNA Class II stimulate dissociation of class I factors from ribosome after release of polypeptide chain. Factors are regulated by GTP binding and hydrolysis 3D structure of RF1 bound to the ribosome

© 2014 Pearson Education, Inc. Figure 15-37a 3D structure of RF1 bound to the ribosome

© 2014 Pearson Education, Inc. Figure 15-37b 3D structure of RF1 bound to the ribosome

© 2014 Pearson Education, Inc. Figure 15-37c 3D structure of RF1 bound to the ribosome

© 2014 Pearson Education, Inc. Figure Comparison of structure of RF1 to a tRNA

© 2014 Pearson Education, Inc. Figure Polypeptide release is catalyzed by two release factors (first through GGQ motif of RF1)

© 2014 Pearson Education, Inc. Polypeptide release is catalyzed by two release factors (first through GGQ motif of RF1) (Class II factor)

© 2014 Pearson Education, Inc. (Class II factor) RFI (Class I factor)

© 2014 Pearson Education, Inc.

Figure RRF (ribosome recycling factor) and EF-G combine to stimulate the release of tRNA and mRNA from a terminated ribosome.

© 2014 Pearson Education, Inc. RRF (ribosome recycling factor) and EF-G combine to stimulate the release of tRNA and mRNA from a terminated ribosome.

© 2014 Pearson Education, Inc. RRF (ribosome recycling factor) and EF-G combine to stimulate the release of tRNA and mRNA from a terminated ribosome.

© 2014 Pearson Education, Inc. Figure Eukaryotic translation termination and ribosome recycling

© 2014 Pearson Education, Inc. Eukaryotic translation termination and ribosome recycling eRF1 recognizes all three stop codons (UAG, UGA, UAA) and bring GGQ motif into peptidyl transferase center leading to peptide release.

© 2014 Pearson Education, Inc. Eukaryotic translation termination and ribosome recycling GGQ motif into peptidyl transferase center leading to peptide release.

© 2014 Pearson Education, Inc. Figure Regulation of bacterial translation initiation by inhibiting 30S subunit binding Protein encoded by the mRNA binds to its RBS. Intramolecular base pairing of the mRNA can interterfere with base pairing by the 16S rRNA.

© 2014 Pearson Education, Inc. Figure 15-42a

© 2014 Pearson Education, Inc. Figure 15-42b Intramolecular base pairing of the mRNA can interterfere with base pairing by the 16S rRNA.

© 2014 Pearson Education, Inc. Box

© 2014 Pearson Education, Inc. Box

© 2014 Pearson Education, Inc. Figure Regulation of translation The protein (ribosomal protein) that acts as a translation repressor of the other proteins, is shaded red.

© 2014 Pearson Education, Inc. Figure rRNA mRNA

© 2014 Pearson Education, Inc. Figure 15-44a

© 2014 Pearson Education, Inc. Figure 15-44b

© 2014 Pearson Education, Inc. Figure Ribosomal protein S8 binds 16S rRNA and its own mRNA Ribosomal protein S8 binds to the region of 16S rRNA Ribosomal protein S8 binds to its own mRNA (S8 mRNA) AUG codon Regulation of translation by binding of S8 to mRNA

© 2014 Pearson Education, Inc. Figure Regulation of eukaryotic translation initiation by eIF4E binding proteins (4E-BPs)

© 2014 Pearson Education, Inc. Figure eIF4E binding protein Cup acts to specifically inhibit Oskar mRNA (in Drosophila oocyte) translation

© 2014 Pearson Education, Inc. Figure (IR(IRE) site E) site (IRE) site Regulation of Ferritin(iron binding protein) translation by iron

© 2014 Pearson Education, Inc. Figure GCN4: transcriptional activator for regulation of gene expression of amino acid biosynthesis enzyme But also regulated at the translational level.

© 2014 Pearson Education, Inc. Figure 15-49a GCN4: transcriptional activator for regulation of gene expression of amino acid biosynthesis enzyme. But also regulated at the translational level. Unlike other eukaryotic ORF, GCN4 has four small ORF (uORF) upstream of the coding sequence eIF2B stimulates eIF2 to exchange GDP for GTP rapidly

© 2014 Pearson Education, Inc. Figure 15-49b eIF2 α kinase (Gcn2) of slow exchange GDP to GTP Slower binding of eIF2-GTP-Met- tRNAi Met to 40S rRNA. So scanning ribosome pass over uORF4 (no binding)

© 2014 Pearson Education, Inc. Figure Translation dependent regulation of mRNA and protein stability: detect defective mRNAs and eliminate them and their products. tmRNA (part of tRNA and part of mRNA) SsrA rescues ribosomes that translate broken mRNA(prematurely terminated mRNA)

© 2014 Pearson Education, Inc. tmRNA (part of tRNA and part of mRNA) SsrA rescues ribosomes that translate broken mRNA(prematurely terminated mRNA)

© 2014 Pearson Education, Inc.

Figure Eukaryotic mRNAs with premature stop codons are targeted degradation. Exon junction complexes (results of splicing) recognize premature stop codon (maybe due to mutation or mistake in transcription) Decapping enzyme Upf proteins activate decapping and deadenylating enzymes

© 2014 Pearson Education, Inc. Exon junction complexes (results of splicing) recognize premature stop codon (maybe due to mutation or mistake in transcription)

© 2014 Pearson Education, Inc. Figure 15-51a

© 2014 Pearson Education, Inc. Figure 15-52

© 2014 Pearson Education, Inc. Non stop mediated decay

© 2014 Pearson Education, Inc. Non stop mediated decay

© 2014 Pearson Education, Inc. No go mediated mRNA decay

© 2014 Pearson Education, Inc. No go mediated mRNA decay