The cap structure, m$ sp7$GpppX (where X = any nucleotide), is present at the 5$ sp prime$ end of all eukaryotic cellular messenger RNAs (except organellar). Previous studies have demonstrated that during protein synthesis the cap structure is recognized by a $ sim$24 kDa cap binding protein (CBP), termed eukaryotic initiation factor 4E (eIF-4E). With the use of a newly developed cap-analogy affinity matrix a high molecular weight complex that contains eIF-4E was purified, termed eIF-4F. In addition to eIF-4E this CBP complex consists of eIF-4A and a 220 kDa polypeptide (p220). Using an RNA unwinding assay direct evidence was obtained indicating that eIF-4F, eIF-4A and eIF-4B functionally interrelate resulting in helicase activity. Secondary structure in the 5$ sp prime$ untranslated region of eukaryotic mRNAs inhibits translation, therefore this unwinding activity is most likely required for efficient 40S ribosomal subunit attachment to mRNA. To fascilitate the purification and biophysical characterization of eIF-4E, the yeast homolog was overexpressed in E. coli. mRNA secondary structure can also inhibit translation in trans by another mechanism. This was shown for the unique structure (TAR) at the 5$ sp prime$ end of all mRNAs from the human immunodeficiency virus-1 (HIV-1). The mechanism of translation inhibition involves the activation of the double-stranded RNA dependent kinase (dsI), which catalyzes the phosphorylation of eIF-2. This is the first demonstration of a specific naturally occurring mRNA sequence that can activate dsI. A novel translational regulatory mechanism is proposed. Finally, the cap structure is also required for efficient precursor mRNA splicing in HeLa nuclear extracts. These and other studies indicate that the cap structure plays a multifunctional role during regulation of gene expression.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.75970 |
| Date | January 1988 |
| Creators | Edery, Isaac |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Biochemistry.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 000913345, proquestno: AAINL52492, Theses scanned by UMI/ProQuest. |
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