Molecular Biology Fourth Edition Chapter 17 The Mechanism of Translation I: Initiation Chapter 18 The Mechanism of Translation II: Elongation and Termination Chapter 19 Ribosomes and Transfer RNA Molecular Biology Fourth Edition Robert F. Weaver

17.1 Initiation of Translation in Bacteria Two important events must occur before translation initiation can take place Generate a supply of aminoacyl-tRNAs Amino acids must be covalently bound to tRNAs Process of bonding tRNA to amino acid is called tRNA charging Dissociation of ribosomes into their two subunits The cell assembles the initiation complex on the small ribosomal subunit The two subunits must separate to make assembly possible

tRNA Charging All tRNAs have same 3 bases at 3’-end (CCA) Terminal adenosine is the target for charging with amino acid Amino acid attached by ester bond between Its carboxyl group 2’- or 3’-hydroxyl group of terminal adenosine of tRNA Amino acid

Two-Step Charging Aminoacyl-tRNA synthetases join amino acids to their cognate tRNAs This is done in a two-step reaction: Begins with activation of the amino acid with AMP derived from ATP In the second step, the energy from the aminoacyl-AMP is used to transfer the amino acid to the tRNA

Aminoacyl-tRNA Synthetase Activity AMP/amino acid coupling AMP/tRNA displacement

Dissociation of Ribosomes E. coli ribosomes dissociate into subunits at the end of each round of translation IF1 actively promotes this dissociation IF3 binds to free 30S subunit and prevents reassociation with 50S subunit to form a whole ribosome

Ribosomal Subunit Exchange Grow in heavy isotope of nitrogen, carbon, and hydrogen. Then 3H labeled

Formation of the 30S Initiation Complex When ribosomes have been dissociated into 50S and 30S subunits, cell builds a complex on the 30S subunit: mRNA Aminoacyl-tRNA Initiation factors IF3 binds by itself to 30S subunit IF1 and IF2 stabilize this binding IF2 can bind alone, but is stabilized with help of IF1 and IF3 IF1 does not bind alone

First Codon and the First Aminoacyl-tRNA Prokaryotic initiation codon is: Usually AUG Can be GUG Rarely UUG Initiating aminoacyl-tRNA is N-formyl-methionyl-tRNA N-formyl-methionine (fMet) is the first amino acid incorporated into a polypeptide This amino acid is frequently removed from the protein during maturation

N-Formyl-Methionine Lipman et al., Marcker and Sanger

Formyl-Met-tRNA and Met-tRNA Which codons they respond to? Which position within the protein they placed methionine?

Which codons they respond to? Make a labeled aminoacyl-tRNA, mix it with ribosomes and a variety of trinucleotides, such as AUG. Met-tRNA bind AUG, formyl-Met-tRNA binds AUG, GUG, and UUG. AUG >90% , GUG about 8% UUG 1%

Which position within the protein they placed methionine? mRNA Sequence AUG AUG AUG……. In vitro translation system In the presence of tRNAmet, met is incorporated into interior of the product In the presence of formy-tRNAmet, met is incorporated into the first codon of the product

Weigert and Garen: Formyl-Met of the the polypeptide is always removed in bacteria and phage proteins

Binding mRNA to the 30S Ribosomal Subunit The 30S initiation complex is formed from a free 30S ribosomal subunit plus mRNA and fMet-tRNA Binding between the 30S prokaryotic ribosomal subunit and the initiation site of a message depends on base pairing between Short RNA sequence Shine-Dalgarno sequence Upstream of initiation codon Complementary sequence 3’-end of 16S RNA

Binding mRNA and fMet-tRNA to the 30S ribosomal subunit How does the cell detect the difference between the initiation codon and an ordinary codon with the same sequences? Consensus sequences?

Positive strand phages f2 MS2 Positive strand phages Encode three genes: A (maturation) protein, coat protein and replicase

Gene structure of MS2 phage RNA

The secondary structures have inhibitory effect

Lodish et al: Translating f2 coat mRNA by ribosomes from different bacteria B. stearothermophilus could only translate A protein but not coat protein (This is not due to initiation factors but ribosomes)

The important element is in the 30S ribosome Nomura et al., The important element is in the 30S ribosome If the 30S ribosome from the E. coli, the coat protein can be translated If the 30S ribosome from the B. stearothermophilus, the coat protein can not be translated The active elements are S12 and 16S rRNA

Shine and Dalgarno Upstream of initiation codon: 5’- AGGAGGU 3’ end of the 16S rRNA: 3’HO-AUUCCUCCAC B. stearothermophilus has poor match

Bacillus 16S rRNA 4 Watson Crick base pairing with the A protein and replicase ribosome binding sites 2 with the coat protein gene E. coli At least three base pairs with all three genes Could the base pairing between 16S rRNA and the region upstream of the translation initiation site be vital to ribosome binding?

Shine-Dalgarno (SD) sequence See Table 17.1 AGGAGGU From ribosomes from C. crescentus and P. aeruginosa No ribosome binding would occur for less than 3 bp

Steitz and Jakes Ribosome from E. coli bound to initiation site and treated with Colicin E3 (RNase) Fingerprinting Initiation site including S-D sequence An oligonucleotide from 3’end of the 16S rRNA

The best evidence Translation Hui and De Boer in 1987 Clone the human growth hormone gene into E. coli Translation WT SD sequence WT 16S rRNA GGAGG CCUCC (5 pairing) OK SD sequence 16S rRNA CCUCC CCUCC (no pairing) NO GUGUG CCUCC (2 pairing) GGAGG (5 pairing)

Initiation Factors and 30S Subunit Binding of the Shine-Dalgarno sequence with the complementary sequence of the 16S rRNA is mediated by IF3 Assisted by IF1 and IF2 All 3 initiation factors have bound to the 30S subunit at this time

Binding of fMet-tRNA to the 30S Initiation Complex IF2 is the major factor promoting binding of fMet-tRNA to the 30S initiation complex Two other initiation factors also play an important supporting role GTP is also required for IF2 binding at physiological IF2 concentrations The GTP is not hydrolyzed in the process

30S Initiation Complex The complete 30S initiation complex contains one each: 30S ribosomal subunit mRNA fMet-tRNA GTP Factors IF1, IF2, IF3

Formation of the 70S Initiation Complex GTP is hydrolyzed after the 50S subunit joins the 30S complex to form the 70S initiation complex This GTP hydrolysis is carried out by IF2 in conjunction with the 50S ribosomal subunit Hydrolysis purpose is to release IF2 and GTP from the complex so polypeptide chain elongation can begin

What is the function of GTP hydrolysis GTP hydrolysis is to remove IF-2 from the ribosomes Exp: 30S initiation complex+labeled IF-2 and fMet-tRNA and either GDPCP or GTP, add 50S and ultracentrifugation

17.18 Effect of GTP hydrolysis on release of IF-2 from the ribosome. After adding 50S, IF-2 is released from the 70S ribosome but fmet-tRNA is still associated

In Fig 17.18, more fMet-tRNA is associated with the 70S ribosomes Catalytic function of IF2 Hydrolysis of GTP is necessary to release IF2 from the 70S initiation complex so it can bind another molecule of fmet-tRNA, otherwise, IF2 only acts stoichiometrically

Bacterial Translation Initiation IF1 influences dissociation of 70S ribosome to 50S and 30S Binding IF3 to 30S, prevents subunit reassociation IF1, IF2, GTP bind alongside IF3 Binding mRNA to fMet-tRNA forming 30S initiation complex Can bind in either order IF2 sponsors fMet-tRNA IF3 sponsors mRNA Binding of 50S with loss of IF1 and IF3 IF2 dissociation and GTP hydrolysis

17.2 Initiation in Eukaryotes Eukaryotic Begins with methionine Initiating tRNA not same as tRNA for interior No Shine-Dalgarno mRNA have caps at 5’end Bacterial N-formyl-methionine Shine-Dalgarno sequence to show ribosomes where to start

Scanning Model of Initiation Eukaryotic 40S ribosomal subunits locate start codon by binding to 5’-cap and scanning downstream to find the 1st AUG in a favorable context Kozak’s Rules are a set of requirements Best context uses A of ACCAUGG as +1: Purine (A or G) in -3 position G in +4 position 5-10% cases ribosomal subunits bypass 1st AUG scanning for more favorable one

The scanning model of initiation Kozak systematically mutated nucleotides around the initiation ↓ codon in a cloned rat preproinsulin gene Introduce into COS cells Label newly synthesized protein with 35S-Met Immunoprecipitate Electrophoresis Detect by autoradipgraph

The scanning model of initiation The better the translation initiation, the more proinsulin was made

The best initiation occur with a G or an A in position –3 and a G in position +4 (where the A in ATG is position +1) A/G C C A T G G -3 -2 -1 +1 +2 +3 +4

Figure 17.21 Effects of single base changes in positions –3 and +4 surrounding the initiating ATG.

If this really is the optimum sequence for translation initiation, introducing it out of frame and upstream of the normal initiation codon should provide a barrier to scanning ribosomes and force them to initiate out of frame

The closer it resembled the optimal sequence, the more it Out-of-frame ATG The closer it resembled the optimal sequence, the more it interfered with initiation at the downstream ATG

Translation With a Short ORF Ribosomes can use a short upstream open reading frame: Initiate at an upstream AUG Translate a short Open Reading Frame Continue scanning Reinitiate at a downstream AUG

Scanning Model for Translation Initiation

Effects of mRNA Secondary Structure Secondary structure near the 5’-end of an mRNA can have either positive or negative effects Hairpin just past an AUG can force a pause by ribosomal subunit and stimulate translation Very stable stem loop between cap and initiation site can block scanning and inhibit translation

Eukaryotic Initiation Factors Bacterial translation initiation requires initiation factors as does eukaryotic initiation of translation Eukaryotic system is more complex than bacterial Scanning process Factors to recognize the 5’-end cap

Translation Initiation in Eukaryotes Eukaryotic initiation factors and general functions: eIF2 binds Met-tRNA to ribosomes eIF2B activates eIF2 replacing its GDP with GTP eIF1 and eIF1A aid in scanning to initiation codon eIF3 binds to 40S ribosomal subunit, inhibits reassociation with 60S subunit eIF4 is a cap-binding protein allowing 40S subunit to bind 5’-end of mRNA eIF5 encourages association between 60S ribosome subunit and 48S complex eIF6 binds to 60S subunit, blocks reassociation with 40S subunit Fig. 17.22

Function of eIF4 eIF4 is a cap-binding protein This protein is composed of 3 parts: eIF4E, 24-kD, has actual cap binding activity eIF4A, a 50-kD polypeptide eIF4G is a 220-kD polypeptide The complex of the three polypeptides together is called eIF4F

The cap-binding proteins GDP-binding protein Cap binding protein

Function of eIF4A and eIF4B Has an RNA helicase activity This activity unwinds hairpins found in the 5’-leaders of eukaryotic mRNA Unwinding activity is ATP dependent eIF4B Has an RNA-binding domain Can stimulate the binding of eIF4A to mRNA

Function of eIF4G eIF4G is an adaptor protein capable of binding to other proteins including: eIF4E, cap-binding protein eIF3, 40S ribosomal subunit-binding protein Pab1p, a poly[A]-binding protein Interacting with these proteins lets eIF4G recruit 40S ribosomal subunits to mRNA and stimulate translation

Inhibit capped cellular mRNA Fig. 17.27 Inhibit capped cellular mRNA Synergy effect Regulatory protein and miRNA bound to the 3’UTR are close to the cap, which could help them influence the cap 2. Ribosomes copleteing one round of translation are close to the cap 3. Unavailable to RNase

Structure and Function of eIF3 eIF3 is a 5-lobed protein that binds at the same site to: eIF4G Prominent part of viral IRES This explains how the IRES can substitute for 40S ribosomal subunit to mRNA Cryo-EM studies have produced a model for the eIF3-IRES-40S complex explaining how eIF3 prevents premature 40S-60S association (sits at the site of proposed key contact between two particles)

Prevention of Premature 40S-60S Association eIF3 blocks key contact point between subunits 40S and 60S eIF4G, so also eIF4E, locate close to the cap on an mRNA bound to 40S ribosomal particle eIF4 would be in position to cap-bind

Functions of eIF1 and eIF1A eIF1 and eIF1A act synergistically to promote formation of a stable 48S complex involving: Initiation factors Met-tRNA 40S ribosomal subunits bound at initiation codon of mRNA eIF1 and eIF1A act by Dissociating improper complexes between 40S subunits and mRNA Encouraging formation of stable 48S complexes

Principle of the Toeprint Assay Source: Adapted from Jackson, R., J. G. Sliciano, Cinderella factors have a ball, Nature 394:830, 1998.

Preformed complex I is not a dead end Marker 17.32

Functions of eIF5 and eIF5B (eIF5 encourages association between 60S ribosome subunit and 48S complex) eIF5B is homologous to prokaryotic factor IF2 Binds GTP Uses GTP hydrolysis to promote its own dissociation from ribosome Permits protein synthesis to begin Stimulates association of 2 ribosomal subunits Differs from IF2 as eIF5B cannot stimulate binding of initiating aminoacyl-tRNA to small ribosomal subunit eIF5B works with eIF5

17.3 Control of Initiation Given the amount of control at the transcriptional and posttranscriptional levels, why control gene expression at translational level? Major advantage = speed New gene products can be produced quickly Simply turn on translation of preexisting mRNA Valuable in eukaryotes Transcripts are relatively long Take correspondingly long time to make Most control of translation happens at the initiation step

Bacterial Translational Control Most bacterial gene expression is controlled at transcription level Majority of bacterial mRNA has a very short lifetime Only 1 to 3 minutes Allows bacteria to respond quickly to changing circumstances Different cistrons on a polycistronic transcript can be translated better than others

Shifts in mRNA Secondary Structure mRNA secondary structure can govern translation initiation Replicase gene of the MS2 class of phages Initiation codon is buried in secondary structure until ribosomes translating the coat gene open up the structure Heat shock sigma factor, s32 of E. coli Repressed by secondary structure that is relaxed by heating Heat can cause an immediate unmasking of initiation codons and burst of synthesis

Proteins/mRNAs Induce mRNA Secondary Structure Shifts Small RNAs with proteins can affect mRNA 2° structure to control translation initiation Riboswitches can be used to control translation initiation via mRNA 2° structure 5’-untranslated region of E. coli thiM mRNA contain a riboswitch This includes an aptamer that binds thiamine and its metabolite Thiamine phosphate Thiamine pyrophosphate (TTP)

Activation of mRNA Translation When TPP abundant Binds aptamer Causes conformational shift in mRNA Ties up Shine-Dalgarno in 2° structure Shift hides the SD sequence from ribosomes Inhibits translation of mRNA Saves energy as thiM mRNA encodes an enzyme needed to produce more thiamine and TPP

Eukaryotic Translational Control Eukaryotic mRNA lifetimes are relatively long, so there is more opportunity for translation control than in bacteria eIF2 a-subunit is a favorite target for translation control Heme-starved reticulocytes activate HCR (heme-controlled repressor) Phosphorylates eIF2a Inhibit initiation Virus-infected cells have another kinase, DAI Inhibits translation initiation

Repression of Translation by Phosphorylation

Phosphorylation of an eIF4E-Binding Protein Insulin and a number of growth factors stimulate a pathway involving a protein kinase known as mTOR mTOR kinase’s target protein PHAS-1 (rat) 4E-BP1 (human) Once phosphorylated by mTOR This protein dissociates from eIF4E Releases it to participate in active translation initiation

Stimulation of Translation by Phosphorylation of PHAS-1

Blockage of Translation Initiation by an miRNA miRNA controls gene expression in two ways: Degradation of mRNA when base-paired perfectly Inhibition of protein production if not perfect match Filipowicz et al conTranslation initiation that is cap-independent due to presence of an IRES, or a tethered initiation factor, is not affected by let-7 miRNA This miRNA blocks binding of eIF4E to the cap of target mRNAs in the human cell

RNA Degradation 80% 20% 10x 10x P1573-

Blockage of Translation Initiation by an miRNA The let-7 miRNA shifts the polysomal profile of target mRNAs in human cells toward smaller polysomes This miRNA blocks translation initiation in human cells Translation initiation that is cap-independent due to presence of an IRES, or a tethered initiation factor, is not affected by let-7 miRNA This miRNA blocks binding of eIF4E to the cap of target mRNAs in the human cell

Fig. 17.46 Collect and fractionate polysomes from cells transfected with RL-3xBulge gene and perform Northern blotting Polysomes: A mRNA attached to several ribosomes (Ch. 19) The more active translation initiation, the more ribosome will attach to the mRNA Anti-miRNA inhibitor revert this effect

Fig. 17.47

Blockage of Translation Initiation by an miRNA Lin-4 miRNA does not alter the polysome profile of its target mRNA in C. elegans and does not appear to block translation initiation. Different miRNAs have different modes-of-action miRNAs work differently in different organisms Or both