Molecular Microbiology (2008) 67(5), 1012–1026 Jae-Ho Yoo† and Uttam L. RajBhandary*

1. Requirements for translation re-initiation in Escherichia coli:roles of initiator tRNA and initiation factors IF2 and IF3 Molecular Microbiology (2008) 67(5), 1012–1026 Jae-Ho Yoo† and Uttam L. RajBhandary* Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA Presentation by: Liz Gallo

2. Introduction Purpose: investigate requirements for translation re-initiation in E.coli by: • constructing a di—cistronic reporter based on the translationally coupled geneV-geneVII pair from M13 phage • studying the effects of using mutant initiator tRNA’s • studying the effects by modulating IF2 and IF3 activity

3. Key Terms • Di-cistronic – translates 2 proteins • Polycistronic – translates many proteins • Intercistronic region – distance between the stop codon of upstream ORF and the start codon of the downstream ORF • Reporter – gene that researchers attach to a regulatory sequence of another gene of interest; often used as an indication of whether a certain gene has been taken up or expressed • Shine-Dalgarno Sequence - is a ribosomal binding site in the mRNA, generally located 8 base pairs upstream of the start codon AUG. (consensus sequence) • M13 phage – circular bacteriophage consisting of ssDNA • GeneV & Gene VII- genes are coupled in M13 phage

4. Abbreviations • CAT – chloramphenicol acetyltransferase • CL – wildtype CAT/fLuc • fLuc – firefly luciferase • am1 – mutant UAG start codon • rbs – SD sequence

5. What are CAT and fLuc??? • CAT – chloramphenicol acetyltransferase • Bacterial enzyme that is responsible for chloramphenicol (broad spectrum antibiotic) resistance in bacteria • fLuc – luciferase • used in bioluminescence, from the tail of a firefly – catalyses the production of light • Can be measured by: RLU/OD and immunoblot assay

6. Background • Three pathways of translation initiation are found in E.coli: • De novo initiation – 30S ribosomal subunit binds to mRNA containing a Shine-Dalgarno (SD) sequence (from scratch) • Re-initiation (see below) • Initiation with leaderless mRNA – mRNA with 0 or very few nucleotides upstream from start codon

7. Polycistronic Operons • Eubacteria contain many genes that are part of polycistronic operons and appear to be coupled • Translational coupling and re-initiation are important for expression of these polycistronic operons

8. Re-initiation • What is re-initiation? • Re-initiation occurs when the same ribosome used to translate an upstream open reading frame (ORF) also translates a downstream ORF. • Couples translation of a downstream gene to translation of an upstream gene aka translational coupling • Little is known about this mechanism

9. The Study • To study translational re-initiation, the intercistronic region from gene V - gene VII from M13 bacteriophage was used to design and construct an inducible, di-cistronic reporter system

10. Di-cistronic reporter • A 72 nucleotide sequence (last 41-13aa of gene V and first 30 – 10 aa of gene VII and a C in-between) was linked downstream of CAT and upstream of fLuc • This represents a di-cistronic operon – used to confirm that both reporters (CAT and fLuc) were co-transcribed and co-translated • fLuc - servers as a reporter for translation re-initiation • CAT –monitors de novo initiation as well as normalizes re-initiation activity to levels of ribosomes that enter the re-initiation site after translating CAT • Under transcriptional control of inducible arabinose promoter

11. Creating Di-cistronic Reporter • Using M13 DNA as a template, a DNA fragment containing the entire geneV and part of geneVII was amplified by PCR • PCR product was digested with restriction enzymes, and cloned • CAT and fLuc also amplified by PCR cut with restriction enzymes and ligated to plasmid • Resulting in: artificial operon encoding CAT_geneV and geneVII_fLuc

12. Di-cistronic reporter system • Confirms that both reporters (CAT and fLuc) were co-transcribed and co-translated • E.coli cells were transformed with WT CL, and used arabinose as the inducer • Cell extracts were analyzed for reporter CAT and fLuc activity and for protein expression level activity • Assays for fLuc activity showed an increase in activity with increasing levels of arabinose • Immunoblot analysis also show an increase in activity with an increase of arabinose

13. How do we know fLuc expression is coupled to CAT? • AUG initiation codons of the CAT and fLUC genes were mutated • Mutant initiator tRNA genes were cloned into reporter plasmids that contained the mutant reporter gene with the corresponding non-AUG start • WT = CL, Mutants = subscripts

14. Results • When start codon in CAT was altered there was no detectable CAT expression and fLuc expression was abolished (lane 3) • When fLuc start codon is mutated expression of full length fLuc was abolished but expression of CAT and internally initiated luciferase fragments were unaffected (lane 4)

15. Intercistronic Distance • Increased intercistronic distance between STOP codon of CAT and START codon of fLuc • SupF – amber (UAG) supressor • Re-initiation generally decreaseswith increased intercistronic distance due to increased probability of ribosomal dissociation

16. Shine-Dalgarno Sequence • To determine if SD sequence would increase translation re-initiation of fLuc a GAGG sequence was inserted 9 nucleotides upstream from the fLuc start codon • SD increased fLuc activity

17. Activity of mutant initiator tRNAs in re-initiation • Changing CAU(wt) anticodon to CUA and GAC allowed the mutant initiator tRNA to base pair with mutant start UAG and GUC codons

18. Comparison of mutant initiator tRNA U35A36 (CUA) in de novo initiation of mutant CAT from Cam1L and re-initiation of mutant fLUC from CLam1

19. Requirements in initiator tRNA for translation re-initiation • Eubacterial initiator tRNAs are different from elongator tRNAs • Met-tRNAfMet fMet-tRNAfMet by MTF • Binding of fMet-tRNAfMet to ribosomal P site • Properties include: • Mismatch at end of acceptor stem for recognition by MTF • 3 consecutive G:C base pairs in the anticodon stem for binding to the ribosomal P-site

20. tRNA Mutants • U35A36/G72G73 (G72/G73) – defective in formylation • C30:G40/U35A36 (C30G40) and U29C30A31:U39G40A41/U35A36 (3GC) – defective in binding of tRNA to ribosomal Psite

21. Requirements in initiator tRNA for translation re-initiation • E.coli were transformed with either Cam1L or CLam1 reporter carrying a mutant initiator tRNA gene • Extracts assayed for CAT and fLuc activity levels

22. Background Continued • E.coli express three essential translation initiation factors that are necessary for efficient and accurate de novo translation initiation •  Initiation factors, mRNA, fMet-tRNAfMet and the 30S ribosomal subunit form the 30S initiation complex (IC) • IF1 – not well studied • IF2 – facilitates binding of fMet-tRNAfMet to the P site of the 30S initiation complex (30S IC) • IF3 – facilitates selection of initiator tRNA and initiation codon by destabilizing 30S ICs that contain non-initiator tRNA or non-canonical codon-anticodon pairing in P site

23. IF2 activity is important for efficient re-initiation • Effects of overproducing IF2 and MetRS on re-initiation of the mutant fLuc reporter • Overproduction increased the re-initiation efficiency of U35A36 mutant tRNA • Overexpression of MetRS leads to increases synthesis of mutant fLuc

24. Mutant initiator tRNA with higher affinity for IF2 is more active in re-initiation • IF2 is required for efficient re-initiation in vivo and re-initiation may have a greater requirement for IF2 than de novo initiation

25. Overexpression of IF3 decreases efficiency of re-initiation • E.coli cells transformed with CL and expression plasmids • Cell extracts were analyzed using immunoblots with anti β-lactamase or anti-CAT Ab (top) or anti-CAT and anti fLuc AB (bottom)

26. SD sequence and IF3 • Inhibitory effects of overexpression of IF3 on re-initiation were less severe when a SD sequence was present

27. Overexpression of IF3 interferes with M13 phage reproduction • Would overproduction of IF3 also reduce the levels of gene VII protein made in cells infected with M13 phage? • E.coli that over produced IF3 were compromised as hosts for M13 • Overproduction of IF3 interferes with a step involved with phage replication/and or assembly, but not adsorption to the cell

28. The Big Picture • Mutant fLuc reporter gene can be translated by re-initiation from non AUG codons • Formylation of aa and tRNA binding to P site are important for re-initiation • IF2 is required for re-initiation, and IF3 also plays a role; overproduction of IF3 seems to inhibit re-initiation

29. Model for translation re-initiation in E. coli

30. PCR Site-Directed Mutagenesis • Was used to create tRNA and mRNA mutants • PCR with olgionucleotide  primers that contain the desired mutation were created. By creating a mutation during the first cycle in binding the template DNA strand, a mutation can be introduced. • After a number of cycles the mutated fragment will be amplified sufficiently to separate from the original, unmutated plasmid by a technique such as gel electrophoresis, and reinstalled in the original context using standard recombinant molecular biology techniques.

31. To Create Mutants – Site Directed Mutagenesis • You need two primers, complementary to each other, containing the new (mutant) sequence flanked by 20 bases on each side. For example, suppose you have the following sequence in some gene in some plasmid CTA CTT CCA GAG ACA ACT GAT CTC TAC TAC TAT GAG CAA TTA AAT GAC AGC GGG • And you want to change it to;CTA CTT CCA GAG ACA ACT GAT CTC TAC TTC TAT GAG CAA TTA AAT GAC AGC GGG • One primer will be;. . . . .5' . . CCA GAG ACA ACT GAT CTC TAC TTC TAT GAG CAA TTA AAT GAC AGC 3' • and the other primer will be the exact complement;. . . . .5' . . GCT GTC ATT TAA TTG CTC ATA GAA GTA GAG ATC AGT TGT CTC TGG 3‘ • Heat the plasmid to separate its strands. • Anneal mutagenic primers that contain the TTC codon, or its reverse complement, GAA. • Perform a few rounds of PCR (about 8) with the mutagenic primers to amplify the plasmid with the altered codon

34. Further Study • Is the 70S ribosome involved in re-initiation?

35. The END!

36. Polymerase Chain Reaction • PCR– method for making many copies of a specific segment of DNA, starting with very small amounts (amplifies DNA) • DNA to be amplified is mixed with DNA oligonucleotides, Taq polymerase, and primers • Mix is heated (break H bonds/sep DNA strands) and cooled (allow DNA primers to anneal) • Primers hybridize to ends of gene to be amplified and provide a starting point for Taq P. which synthesizes complementary strands of DNA • Go through “thermal cycling” –until enough DNA has been produced

38. Transformation • Add transformation solution (ex CaCl2) to tube • Place on ice and add E.coli colony • Add fragment containing gene(s) of interest (incubate on ice) • Heat shock – place tube into a warm heat bath for a little under a minute, place back on ice (about 2 minutes) • Add nutrients (LB nutrient broth) and sit at room temp (10 minutes) • Transfer to agar plates (these plates contain ampicillin selection and mutants require arabinose inducer)