1. Messenger RNA
Todd Metzger
BMS 265
May 8, 2009

2. Nomenclature Messenger RNAs (mRNA) describes cytoplasmic product
Pre-mRNA (part of hnRNA) in nucleus is processed and exported
Describe by gene/protein, including isoform
or NCBI sequence (NM, natural; XM, in silico)
New methods for describing spliced mRNAs (Sammeth 2008)

3. 1955 - Goldstein and Plaut 6 min 62 hr
Radiolabeled RNA, transferred nucleus to new amoeba, observed movement to cytosol over time
62 hr

4. Discovery
Volkin and Astrachan discovered a new type of RNA synthesized following infection of bacteria by T2 Bacteriophage (Volkin 1956)
Newly synthesized RNA mirrored composition of T2 DNA
Brenner, Jacob, and Meselson found that only RNA, and not ribosomes, were newly synthesized following infection (1961)
The term messenger RNA was coined
New material that led to bacteriophage production must be carried on RNA and not on ribosomes

5. Translation Leads to Linear Protein Synthesis
Dintzis 1961
Reticulocytes analyzed
Make ~90% hemoglobin
Radiolabeled amino acids incorporated to C-terminus only

6. 1970 - Messenger RNA Visualized Miller et al
EM images showing coupled transcription and translation in E. coli

7. Biogenesis Enzymatic Reaction by RNA Polymerase
RNA Pol II in eukaryotes
Involves Initiation Factors (TFIIB,D,E,F,H) and Mediator Complex
Core RNA polymerase II is 12 subunits, 500 kDA, highly conserved
10 subunit catalytic core
Woychik 2002, Sims III 2004

8. Yeast RNA pol II Catalytic core Positively charged cleft
RNA pol II structure can allow proofreading
Active site occurs between the wall and clamp (shown closed - elongation)

9. Transcription
Template DNA, 5 GTFs, and RNA pol II associated to form pre-initiation complex
TFIID contains the TATA-box binding protein (TBP)
TFIIE and -H melt DNA
CTD phosphorylation is required for dissociation of initiation factors and entrance to the elongation phase
TFIIS allows stalled transcription to continue
Reverse procession followed by cleaved of RNA

10. TFIIS
Could participate in proofreading

11. Mediator Connects transcription factors to basal machinery
Thought to stabilize and promote initiation
Casamassimi 2007

13. mRNA Secondary Structure
Many mRNAs can code for the same protein
Made program to scramble codons while preserving base content
Looked at 50 bp structures
redundancy
Katz 2003

14. mRNA Secondary Structure
- No significant differences for human sequences

15. mRNA Vaccinations - Direct
Ex vivo DC transfection known to work
Hess et al 2006

16. mRNA Vaccinations - Indirect
Ex vivo DC transfection known to work
Hess et al 2006