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Gene Expression MBIOL 6640 10:45-11:35 ASB 210 Course director: Dean Tantin, PhD X7-3035, [email protected] Lecture 0 For many of you this is a review of undergraduate material, but study these slides in advance of the first day of class. There is much that is testable in here, and things in lectures 14 will blow by you if you are not familiar with this material. (Simplified) flow of genetic information… transcription DNA pre-mRNA mRNA splicing Protein translation Gene expression begins with the process of TRANSCRIPTION Some terminology (applies to prok. and euk. transcription): • Transcription is the synthesis of an RNA chain from a DNA template. • RNA synthesis proceeds in the 5’3’ direction and is performed by RNA polymerase and ancillary factors. • RNA synthesis utilizes NTPs, with uridine triphosphate (UTP) in place of dTTP in base pairing with adenosine. Thus, RNA is marked by the presence of a 2’ hydroxyl and uridylate. • Only one strand of the DNA double helix is utilized as the physical template for RNA synthesis, and is termed the template strand. • The coding strand has a 5’-3’ sequence that is identical to the 5’3’ sequence of the RNA. Gene expression begins with the process of TRANSCRIPTION More terminology: • The promoter is a region of DNA at the 5’ end of the gene that controls its expression. In eukaryotes it includes the initiation site (start site), the first base that is transcribed (bacteria=operator). • The terminator is a DNA sequence that directs where RNA synthesis stops (this is different from a stop codon). The intervening DNA is considered the “body” of the gene. A transcription unit is all RNA synthesized in a single RNA chain. An “operon” is a bacterial transcription unit that consists of multiple open reading frames coding for multiple proteins. • The standard nomenclature for describing the position of DNA base pairs relative to the start site considers +1 as the start site and -1 as the base before it (there is no zero). Downstream DNA is any DNA that is found in the direction the RNA polymerase travels. The opposite is true for upstream DNA. Gene expression begins with the process of TRANSCRIPTION Overview of the prokaryotic transcription cycle The basic form of the cycle is conserved in eukaryotes. Promoter (template) recognition: Core RNA polymerase cannot find a promoter by itself. It requires promoter specificity factors, called sigma (s) factors. Open complex formation and initiation: --The DNA is unwound to form the bubble. --Formation of the first phopshodiester bonds. 2-9 base “abortive transcripts” synthesized. Promoter clearance (promoter escape): The transition to processive transcription; the bubble travels with the polymerase. Elongation: Incorporation of ribonucleotides into the RNA chain. Termination: Synthesis ends and the RNA is released. Promoter recognition—bacterial RNA polymerase Two forms: • ‘Core’ with the subunit composition aabb’w (=a2bb’w). • ‘Holoenzyme’* also includes s. • There are multiple s factors, so the polymerase can recognize promoters with different recognition sequences. • The most common s factor is s70. Eukaryotes lack true sigma factors, and have different strategies for recognizing promoters. *‘Holoenzyme’ refers to an active form in which all the subunits and cofactors needed for activity are present (as contrasted with ‘apoenzyme’). Promoter recognition—bacterial RNA polymerase b’—largest subunit. Forms part of active site. Interacts nonspecifically with DNA and nascent RNA. b—forms the rest of the active site. Interacts nonspecifically with DNA and nascent RNA. a—determinants for assembly of RNAP. Recognizes DNA nonspecifically, OR can recognize a specific Upstream Promoter element (“UP element”) at certain highly expressed bacterial genes. w—stabilizes the assembled RNAP. Sigma factors greatly increase the affinity of polymerase for promoter DNA (by decreasing the off rate). Holoenzyme bound to DNA that is unmelted is called the closed complex.