1. Translation initiation mediated by RNA looping
Presented by
Chris & Desmond
Ki Young Paeka, Ka Young Honga, Incheol Ryua, Sung Mi Parka, Sun Ju Keuma, Oh Sung Kwona, and Sung Key Janga,b,
Ferguson

2. Overview Mechanism of Translation Initiation in Eukaryotes
eIF4F complex and functions
Cap-dependent and IRES mediated Translation Initiation
Alternative mechanisms to the classical scanning model
Techniques
Reporter Assays
Co - immunoprecipitation assays
In Vitro Translation
Other Information
G-cap
Kuhn Length

3. Translation Initiation of Eukaryotic

4. eIF4F complex
eIF4G - eIF4G serves as a scaffold protein for the assembly of eIF4E and eIF4A and other proteins.
eIF4A - eIF4A is an ATP-dependent and has RNA helicase activity (catalyze the unwinding of mRNA secondary structure at the 5'UTR).
eIF4E - eIF4E is a cap-binding factor.

5. Translation Initiation of Eukaryotic
Cap-dependent translation initiation
IRES Mediated Translation Initiation

6. Alternative mechanisms to the classical scanning model
Leaky scanning. The 40S ribosomal subunit can either recognize the start codon or it can scan past the upstream start codon and initiate translation at a downstream start codon.
Ribosomal shunt. 40S jumps over the secondary structure or upstream initiation codons.
Ribosomal re-initiation. This a process which allows expression of a downstream ORF in a bicistronic mRNA.

7. Techniques
Dual-Reporter Assay
Co - immunoprecipitation
In-Vitro Translation

8. Dual-Reporter Assay Benefits Greater Accuracy Convenience
The system relies on two different reporter genes within a system.
Benefits
Greater Accuracy
Convenience
Sensitivity: Allows study of weak promoters, low-level expression/regulation and expression in cells that transfect poorly.

9. The Dual-Luciferase® Reporter (DLR™) Assay System
Why Two Reporters?
The dual measurement of these two enzymes allows for an experimental measurement and a transfection control measurement to be done at the same time. This dual reporting of each sample allows a quantitative result based on the normalization of the Renilla luciferase (transfection control).

10. Co-Immunoprecipitation

11. In Vitro Translation Standard Translation Systems u
Linked Transcription: Translation u

12. G-cap
Adding a G-cap typically significantly inhibits 5’ end-dependent translation.
When G-cap does not significantly inhibit translation the mechanism might be 5’ independen.t (As in RNA Looping).

13. Random Walk
A random walk is a mathematical formalization of a path that consists of a succession of random steps (at random angles)
Image is of a 2 dimensional
random walk

14. Kuhn Length
Developed by Werner Kuhn to make sense of polymer’s (3D) random walk: Successive rigid molecular segments added at a random angle with freely jointed ends.
Used as a theoretical treatment to describe an “ideal” chain as opposed to a “real” chain. (Used for 5’ UTR of mRNA in this experiment)
Having a single nucleotide as segment would greatly underestimate end-to-end distance. Segments therefore modeled as many nucleotides in length.
Kuhn length represents average length (in # of nucleotides) assigned to a segment.
Kuhn length is longer for stiffer molecules.
Info from:

15. Kuhn Length
Equation predicting probable density (jM) of two sites separated by a distance (n) in a linear polymer jM = local concentration n= # of Kuhn segments I= statistical Kuhn segment length (in nanometers) d= special parameter (0 in this experiment)

16. A: Relative translation efficiencies of mRNA’s with 5’UTR of varying length (Experimental). Peak at 70 nts
B: Relationship between local concentration and distance in # of Kuhn segments (Theoretical). Peak at n = 1.62
# of Kuhn segments (n) = = 70 nt
Kuhn length (I) = 70/1.62 = 43.8 nucleotides.
Kuhn length (I) = 43.8 nt X .65nm/1nt = nanometers

17. Correlation between predicted local concentration (jM in equation) with experimental translation efficiency.
Authors claimed strong linear correlation indicating governance of RNA looping over translation initiation.
R2 of 0.76 leaves a lot to be desired.