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

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

Translation Initiation of Eukaryotic

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.

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

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.

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

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.

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).

Co-Immunoprecipitation

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

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).

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

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: http://www.doitpoms.ac.uk/tlplib/polymerbasics/morphology.php

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)

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) = 1.62 = 70 nt Kuhn length (I) = 70/1.62 = 43.8 nucleotides. Kuhn length (I) = 43.8 nt X .65nm/1nt = 28.47 nanometers

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.