Transcript No Slide Title

Signposts for translation initiation:
An illustration of formulating a
research project
Xuhua Xia
[email protected]
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
What is known:
50
• From “reading”:
45
•
Signposts for translation initiation are often
located around or upstream of the start
codon.
•
The signposts are often a short motif
40
% Frequency
35
• From “doing”: a dramatic pattern
30
A
C
25
G
U
20
15
10
5
0
10
20
30
40
50
Site
60
70
80
90
100
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
55
55
Highly expressed genes
Lowly expressed genes
45
35
A
C
G
U
% Frequency
% Frequency
45
35
A
C
G
U
25
25
15
15
5
5
0
20
40
60
Site
80
100
0
20
40
60
80
100
Site
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
ASD: 3’ AUUCCUCCACUA---..5’
SD: 5..--AGGAGG---..AUG–..3’
Secondary structure of E. coli 16S rRNA
Yassin A et al. PNAS 2005;102:16620-16625
Modern Definition of SD
SD1
SD2
mRNA
D2
A U G
aSD: pyrimidine-rich
DtoAUG
ssu Ribosome
Number of SD-aSD pairs
(c)
D1
(a)
800
600
400
200
0
0
10
20
30
40
DtoAUG
(d)
Number of hits
(b)
600
400
200
0
Is it important to have weak bonds here so
that the stem can be open a bit to increase
flexibility?
AUUCCUCCACUAG
3' tail of SSU rRNA
Prabhakaran et al. 2015
Refine the hypothesis
16S rRNA
Z2705
3’ ATTCCTCCACTAGGTTGGCG--- 5’
GAGATTAACTCAATCTAGAGGGTATTAATAATG
DtoAUG = 17
16S rRNA
Z5748
3’ ATTCCTCCACTAGGTTGGCG--- 5’
CTGAACATACGAATTTAAGGAATAAAGATAATG
DtoAUG = 15
16S rRNA
Z3810
3’ ATTCCTCCACTAGGTTGGCG--- 5’
AACCGCCGCTTACCAGCAGGAGGTGATGAAAUG
DtoAUG = 15
16S rRNA
Z2225
3’ ATTCCTCCACTAGGTTGGCG--- 5’
TGATCCGCGTATCGGACGTGGAGGTGGTGAATG
DtoAUG = 14
It is the pairing, not the motif AGGAGG, that is important.
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
Refine hypothesis to
accommodate new observations
Universally accepted:
Working theory
New observations
contradicting the theory
New hypothesis to
accommodate new observations
Effect of a single substitution
E. coli
GGAUCACCUCCUUA 3’
B. subtilis UCACCUCCUUUCUA 3’
N
E. coli
AUGAUG
500
450
400
350
300
250
200
150
100
50
0
DtoAUG
E. coli
0
2
4
6
8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
DtoAUG
1000
900
800
B. subtilis
700
600
N
B. subtilis
500
400
AUG
300
200
100
DtoAUG
0
0
2
4
6
8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
DtoAUG
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
Probhakaran et al. unpublished.
Xuhua Xia
Slide 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.
Yeast 18S rRNA
people.biochem.umass.edu
Yeast 5’ UTR
70
60
50
Percentage
A
40
30
U
C
20
G
10
0
-150 -140 -130 -120 -110 -100
-90
-80
-70
-60
-50
-40
-30
-20
-10
Site (5'-UTR)
Xuhua Xia
Slide 18
Gene expression and 5’ UTR
Low-Expression Group
High-Expression Group
G
C
A
U
80
80
70
70
60
60
50
50
Percentage
Percentage
A
40
C
U
40
30
30
20
20
10
10
0
G
0
-60
-50
-40
-30
Site
Xuhua Xia
-20
-10
0
-60
-50
-40
-30
Site
-20
-10
0