signposts for translation initiation: an illustration of formulating a research project xuhua xia...
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Signposts for translation initiation:An illustration of formulating a
research project
Xuhua Xia
http://dambe.bio.uottawa.ca
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?– .....
E. coli 5’ UTR
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0 10 20 30 40 50 60 70 80 90 100
Site
% F
req
ue
nc
y
A
C
G
U
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
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)
Results testing the predictions
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0 20 40 60 80 100
Site
% F
req
ue
ncy A
C
G
U
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0 20 40 60 80 100
Site
% F
req
ue
ncy A
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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
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?– Outdated:
• 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).
– Modern
Secondary structure of E. coli 16S rRNAYassin A et al. PNAS 2005;102:16620-16625
ASD: 3’ AUUCCUCCACUA---..5’SD: 5..--AGGAGG---..AUG–..3’
D2
0 5 10 15 20 25 30 35 400
200
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800
DtoAUG
Num
ber
of S
D-a
SD
pai
rsA U U C C U C C A C U A G
0
200
400
600
3' tail of SSU rRNA
Num
ber
of h
its
aSD: pyrimidine-rich
SD1 A U G
DtoAUG
mRNA
ssu Ribosome
SD2
D1(a)
(b)
(c)
(d)
Modern Definition of SD
Is it important to have weak bonds here so that the stem can be open a bit to increase flexibility?
Prabhakaran et al. 2015
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.
DtoAUG = 17
DtoAUG = 15
DtoAUG = 15
DtoAUG = 14
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)
• What genes need SD (still an unanswered question)?
Progress of science
Observation
Hypothesis
Predictions and tests
Universally accepted:Working theory
New observationscontradicting the theory
Refine hypothesis to accommodate new observations
New hypothesis to accommodate new observations
DtoAUG
AUG
E. coli GGAUCACCUCCUUA 3’B. subtilis UCACCUCCUUUCUA 3’
AUGAUG
DtoAUG
0
100
200
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500
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1000
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
DtoAUG
N
0
50100
150
200250
300
350
400450
500
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
DtoAUG
N
E. coli
B. subtilis
E. coli
B. subtilis
Effect of a single substitution
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.
• Secondary structure may embed SD or start codon and hide the translation start signal
• Prediction: reduced secondary structure in seuqences flanking SD and start codon
Secondary structure and start codon
Xuhua Xia Slide 15
Probhakaran et al. unpublished.
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.
Yeast 18S rRNA
people.biochem.umass.edu
Yeast 5’ UTR
Xuhua Xia Slide 18
0
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-150 -140 -130 -120 -110 -100 -90 -80 -70 -60 -50 -40 -30 -20 -10
Site (5'-UTR)
Pe
rce
nta
ge
A
U
C
G
Gene expression and 5’ UTR
Xuhua Xia
Low-Expression Group
0
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-60 -50 -40 -30 -20 -10 0
Site
Per
cen
tag
e
A G C U
High-Expression Group
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-60 -50 -40 -30 -20 -10 0
Site
Per
cen
tag
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A G C U