1 / 19

Signposts for translation initiation: An illustration of formulating a research project

Signposts for translation initiation: An illustration of formulating a research project. Xuhua Xia xxia@uottawa.ca http://dambe.bio.uottawa.ca. The Protocol. What is known (which involves much reading and doing) Formulating hypothesis based on what is known

roxy
Download Presentation

Signposts for translation initiation: An illustration of formulating a research project

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Signposts for translation initiation:An illustration of formulating a research project Xuhua Xia xxia@uottawa.ca http://dambe.bio.uottawa.ca

  2. The Protocol • What is known (which involves much reading and doing) • Formulating hypothesis based on what is known • Derive predictions from the hypothesis: • Predictions are always about the relationship between or among measurable variables. • Predictions involving variables that cannot be measured is of no value. • Design experiments to test the predictions • Methods to measure the variables relevant to the prediction • Methods to assess the relationship among the variables to confirm or reject the predictions • Results • All results should be presented with respect to the predictions. • Anything that is biologically interesting but not directly related to the predictions should be in the Discussion section • Discussion • Does the method measure the variables as you intend it to? • Does your conclusions depend on assumptions that may not be valid under certain circumstances? • .....

  3. E. coli 5’ UTR • What is known: • From “reading”: signposts for translation initiation should be located at the initiation codon and the sequence upstream of the initiation codon • From “doing”: a dramatic pattern

  4. Hypothesis, prediction & methods • Hypothesis: the pattern is related to translation initiation, i.e., a dramatic increase in G and dramatic decreases in C and U enhance translation initiation. • Prediction: If the hypothesis is correct, then we expect highly expressed genes to exhibit the pattern more strongly than the lowly expressed genes. • It is a relationship involving two variables • The gene expression • The strength of the pattern • The variables need to be measurable • Methods: how should we measure the variables? • Gene expression (CAI or wet lab measurement?) • The pattern: graphic characterization

  5. Results testing the predictions Highly expressed genes Lowly expressed genes You could do statistics to show that the pattern in the left is significantly stronger than that in the right, but often a picture is worth 1000 words + 10 p values. Results not directly related to the prediction but should be discussed: the difference in frequency distribution at sites 0-70

  6. Prokaryotic translation initiation • Shine-Dalgarno (SD) sequence in the 5’ UTR matches the anti-SD (ASD) sequence at the 3’ end of ssu rRNA • SD consensus is AGGAGG, binding to UCCUCC in the 3’ end of ssu rRNA • In E. coli, for example, the sequence is AGGAGGU. This sequence helps recruit the ribosome to the mRNA to initiate protein synthesis by aligning it with the start codon. The complementary sequence (UUCCUCC),

  7. ASD: 3’ AUUCCUCCACUA---..5’SD: 5..--AGGAGG---..AUG–..3’ Secondary structure of E. coli 16S rRNAYassin A et al. PNAS 2005;102:16620-16625

  8. Refine the hypothesis 16S rRNA 3’ ATTCCTCCACTAGGTTGGCG--- 5’Z2705 GAGATTAACTCAATCTAGAGGGTATTAATAATG 16S rRNA 3’ ATTCCTCCACTAGGTTGGCG--- 5’Z5748 CTGAACATACGAATTTAAGGAATAAAGATAATG 16S rRNA 3’ ATTCCTCCACTAGGTTGGCG--- 5’Z3810 AACCGCCGCTTACCAGCAGGAGGTGATGAAAUG 16S rRNA 3’ ATTCCTCCACTAGGTTGGCG--- 5’Z2225 TGATCCGCGTATCGGACGTGGAGGTGGTGAATG It is the pairing, not the motif AGGAGG, that is important.

  9. Pairing and reading frame 16S rRNA 3’ ATTCCTCCACTAGGTTGGCG--- 5’Z4094 CAGTTTAACTAGTGACTTGAGGAAAACCTAATG 16S rRNA 3’ ATTCCTCCACTAGGTTGGCG--- 5’Z4094 CAGTTTAACTAGTGACTTGAGGAAAACCTAATG 16S rRNA 3’ ATTCCTCCACTAGGTTGGCG--- 5’Z4981 GGCACACTTAATTATTAAAGGTAATACACTATG 16S rRNA 3’ ATTCCTCCACTAGGTTGGCG--- 5’Z4981 GGCACACTTAATTATTAAAGGTAATACACTATG 16S rRNA 3’ ATTCCTCCACTAGGTTGGCG--- 5’Z0849 TATTAGATTTGTATTCACCGGAGTGATGTAATG 16S rRNA 3’ ATTCCTCCACTAGGTTGGCG--- 5’Z0849 TATTAGATTTGTATTCACCGGAGTGATGTAATG 16S rRNA 3’ ATTCCTCCACTAGGTTGGCG--- 5’Z0749 CATCTCATCGAAAACACGGAGGAAGTATAGATG 16S rRNA 3’ ATTCCTCCACTAGGTTGGCG--- 5’Z0749 CATCTCATCGAAAACACGGAGGAAGTATAGATG Multiple SD sequences binds to 16S rRNA 3n nucleotides apart. Hypothesis: the pairing contributes to the determination of the reading frame Prediction: highly expressed genes should exhibit the pattern more strongly than lowly expressed genes

  10. Hypothesis, prediction, tests • Pairing between SD sequence and ASD are essential for translation initiation • Prediction: Modifying the SD or ASD to disrupt base pairing will reduce protein production • The prediction was initially supported (A. Hui, H. de Boer. 1987. PNAS 84:4762–4766 • Mutating SD to disrupt the pairing: Protein production decreased • Mutating ASD to restore the pairing: Protein production is restored. • Many counter examples (SD not needed for initiation): • The classic Nirenberg and Matthaei experiment with poly-U • P. Melancon et al. 1990. The anti-Shine–Dalgarno region in Escherichia coli 16S ribosomal RNA is not essential for the correct selection of translational starts. Biochemistry, 29:3402–3407 (Removed the last ~30 nt in 16S rRNA) • D.C. Fargo et al. 1998 Shine–Dalgarno-like sequences are not required for translation of chloroplast mRNAs in Chlamydomonas reinhardtii chloroplasts or in Escherichia coli Mol. Gen. Genet. 257:271–282 • S. Sartorius-Neef, F. Pfeifer. 2004 In vivo studies on putative Shine–Dalgarno Sequences of the halophilic archaeon Halobacterium salinarum Mol. Microbiol., 51:579–588 (Efficient translation of leaderless mRNA)

  11. Progress of science Observation Hypothesis Predictions and tests Refine hypothesis to accommodate new observations New hypothesis to accommodate new observations Universally accepted: Working theory New observations contradicting the theory

  12. mRNA AUG SD: purine-rich ASD: pyrimidine-rich DtoAUG ssu Ribosome Z2705 GAGATTAACTCAATCTAGAGGGTATTAATAATG16S rRNA 3’ ATTCCTCCACTAGGTTGGCG--- 5’ DtoAUG= 17

  13. Effect of a single substitution E. coli GGAUCACCUCCUUA 3’ B. subtilis UCACCUCCUUUCUA 3’ E. coli E. coli AUGAUG DtoAUG B. subtilis B. subtilis AUG DtoAUG

  14. A new hypothesis • An accessible initiation codon is essential for translation initiation (T. Nakamoto 2006 BBRC 341:675-678): • A leaderless mRNA can be translated because the initiation AUG is highly accessible at the 5’ end • SD and ASD pairing prevents secondary structure formation involving the initiation AUG and makes the AUG more accessible. • Synthetic mRNA without the SD sequence but can be efficiently translated are typically without secondary structure, rendering the initiation AUG readily accessible.

  15. Another new hypothesis • Translation initiation of both prokaryotic and eukaryotic genes depends on the ssu ribosome scanning along the mRNA • Any mechanism that can pause the ssu ribosome near the initiation codon can enhance translation initiation.

  16. Yeast 18S rRNA people.biochem.umass.edu

  17. Yeast 5’ UTR Slide 18 Xuhua Xia

  18. Gene expression and 5’ UTR Slide 19 Xuhua Xia

More Related