1. Translation initiation and co-evolution between SD and aSD in bacteria
Xuhua Xia

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 in science.
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 are often located around or upstream of the start codon.
The signposts are often a short motif
From “doing”: a dramatic pattern

4. Hypothesis, prediction & methods
Hypothesis: the pattern is related to translation initiation, i.e., a dramatic increase in purine and dramatic decreases in pyrimidine 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 results from wet lab measurements)
The pattern:
graphic characterization
Numerical characterization (e.g., the variance among the four frequencies)

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. Going forward
There is strong signal within ~20 nt upstream of the start codon, likely in the form of short motif.
What motif is it? How does it help translation? Where should it be optimally positioned?
Xuhua Xia

7. lacI gene in E. coli -35 -10 stop codon of the upstream mhpR
----|----|----|----|----|----|----|----|----|----|----|----|----|----|----|----|----|----|
lacI CGAAGCGGCAUGCAUUUACGUUGACACCAUCGAAUGGCGCAAAACCUUUCGCGGUAUGGCAUGAUAGCGCCCGGAAGAGAGUCAAUUCAG
lacI GGUGGUGAAUGUGAAACCAGUAACGUUAUACGAUGUCGCAGAGUAUGCCGGUGUCUCUUAUCAGACCGUUUCCCGCGUGGUGAACCAGGC
......
lacI GCAGCUGGCACGACAGGUUUCCCGACUGGAAAGCGGGCAGUGAGCGCAACGCAAUUAAUGUGAGUUAGCUCACUCAUUAGGCACCCCAGG
----|----|----|----|----|----|----|----|----|----|---
lacI CUUUACACUUUAUGCUUCCGGCUCGUAUGUUGUGUGGAAUUGUGAGCGGAUAA
stop codon of the upstream mhpR
transcription start site SD
start codon
stop codon
ssu rRNA --GAUCACCUCCUUA 3'
mRNA GUGGUGGGA---- 5'
Frequent overlap between stop codon and start codon (of the downstream gene)
---UAAUG---, ---AUGA---

8. aSD: 3’ AUUCCUCCACUA----..5’
SD: AGGAGG---..AUG–..3’
Secondary structure of E. coli 16S rRNA Yassin A et al. PNAS 2005;102:

9. Prokaryotic translation initiation
Shine-Dalgarno (SD) sequence in the 5’ UTR matches the anti-SD (ASD) sequence at the 3’ end of ssu rRNA
What is an SD?
SD consensus is AGGAGG, binding to UCCUCC in the 3’ end of ssu rRNA. This sequence helps recruit the ribosome to the mRNA to initiate protein synthesis by aligning it with the start codon.
Prediction: Modifying the SD or aSD to disrupt base pairing will reduce protein production
The prediction was supported (A. Hui, H. de Boer PNAS 84:4762–4766
Mutating SD to disrupt the pairing: Protein production decreased
Mutating ASD to restore the pairing: Protein production is restored.
Modern definition of an SD:
Any stretch of nucleotides that can base-pair with the 3' end of ssu rRNA to properly position the tRNA anticodon against the start codon.

10. Modern Definition of SD
aSD: pyrimidine-rich
SD1
A U G
DtoAUG
mRNA
ssu Ribosome
SD2 D1
(a)
(b)
(c)
(d)
Fig. 1. Schematic representation of Shine-Dalgarno (SD) sequence on mRNA pairing with anti-SD (aSD) sequence on the small subunit (SSU) rRNA (a). Also drawn are the free 3' end of SSU rRNA (b), the frequency distribution of 4577 putative matches of at least four bases between the 3' tail of rRNA and the upstream 30 nucleotides of CDSs (c), and the number of times each nucleotide sites at 3' tail of rRNA participated in the SD-aSD matches (d).
Is it important to have weak bonds here so that the stem can be open a bit to increase flexibility?
Prabhakaran et al. 2015

11. A sample of SDs
16S rRNA ’ ATTCCTCCACTAGGTTGGCG--- 5’ Z GAGATTAACTCAATCTAGAGGGTATTAATAATG
16S rRNA ’ ATTCCTCCACTAGGTTGGCG--- 5’ Z CTGAACATACGAATTTAAGGAATAAAGATAATG
16S rRNA ’ ATTCCTCCACTAGGTTGGCG--- 5’ Z AACCGCCGCTTACCAGCAGGAGGTGATGAAAUG
16S rRNA ’ ATTCCTCCACTAGGTTGGCG--- 5’ Z TGATCCGCGTATCGGACGTGGAGGTGGTGAATG
DtoAUG = 17
DtoAUG = 15
DtoAUG = 15
Multiple SD sequences bind to 16S rRNA exactly 3 nucleotides apart.
DtoAUG = 14
It is the pairing, not the motif AGGAGG, that is important.

12. Effect of a deletion E. coli GGUCACCUCCUU---A 3’
B. subtilis UCACCUCCUUUCUA 3’
SD:-AGAAAC AUG |||||
aSD:AUCUUUCCUCCACU----
SD:-----AGGAGG AUG ||||||
aSD:AUCUUUCCUCCACU----
SD:-AGAAAGGA AUG ||||||||
aSD:AUCUUUCCUCCACU----
DtoAUG = 18
DtoAUG = 18
DtoAUG = 18
SD:-AGAAAC AUG ||
aSD: AUUCCUCCACU----
SD:-----AGGAGG AUG ||||||
aSD: AUUCCUCCACU----
SD:-AGAAAGGA AUG |||||
aSD: AUUCCUCCACU----
DtoAUG = 15
DtoAUG = 15
DtoAUG = 15
A 3-nt deletion (UCU) would select against AGA-containing SDs
E. coli
B. subtilis

13. Effect of a deletion SD:-AGGGAC-----------AUG |||||
aSD:AUCUUUCCUCCACU----
SD: -AGGGAC augAUG |||||
aSD: AUUCCUCCACU----
Xuhua Xia
Xuhua Xia
Slide 14

14. Hypothesis, prediction, tests
Many counter examples (SD not needed for initiation):
The classic Nirenberg and Matthaei experiment with poly-U
P. Melancon et al 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 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 In vivo studies on putative Shine–Dalgarno Sequences of the halophilic archaeon Halobacterium salinarum Mol. Microbiol., 51:579–588 (Efficient translation of leaderless mRNA)
What genes need SD (still an unanswered question)?
The removal of the last ~30 nt in 16 rRNA lead to reduced protein production, but translation initiation is still at the same initiation codon.
Problem: The removal will create another unpaired 3’ tail that can form base pairs with the 5’ UTR or mRNA (Cf: secondary structure of 16S rRNA).

15. 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

16. A new hypothesis
An accessible initiation codon is essential for translation initiation (T. Nakamoto 2006 BBRC 341: ):
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.
Secondary structure may embed SD or start codon and hide the translation start signal
Prediction: reduced secondary structure in sequences flanking SD and start codon

17. Secondary structure and start codon
Probhakaran et al. unpublished.
Xuhua Xia