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Colinearity of Gene and Protein genotype DNA DNA sequence transcription RNA translation protein function phenotype organism amino acid sequence Genetic Code Genetic Code is nonoverlapping. A codon (three bases or triplet) encodes an amino acid. Genetic Code is read continuously from a fixed starting point. There is a start codon (AUG). There are three stop (termination) codons. They are often called nonsense codons. Genetic Code is degenerate. Some amino acids are encoded by more than one codon. Molecular Basis for Relationship between Genotype and Phenotype genotype DNA DNA sequence transcription RNA translation protein function phenotype organism amino acid sequence Eukaryotic RNA Three RNA Polymerases RNA Polymerase Synthesis of I II III rRNA (except 5S rRNA) mRNA*, some snRNA tRNA, some snRNA, 5S rRNA * eukaryotic RNA is monocistronic prokaryotic RNA can be polycistronic Eukaryotic RNA Many proteins must assemble at promoter before transcription. General transcription factors (GTF’s) bind before RNA polymerase II, while other proteins bind after RNA polymerase II binds. Primary transcript (pre-mRNA) must be processed into mature mRNA. Chromatin structure affects gene expression (gene transcription) in eukaryotes. 1. Cap at 5’ end (7-methylguanosine) 2. Addition of poly(A) tail 3. Splicing of RNA transcript Prokaryotic and Eukaryotic Transcription and Translation Compared Transcription Initiation in Eukaryotes TATA binding protein (TBP), part of TFIID complex, must bind to promoter before other GTFs and RNA polymerase II can form preinitiation complex (PIC). Phosphorylation of carboxyl tail domain (CTD), the protein tail of b subunit of RNA polymerase II, allows separation of RNA polymerase II from GTFs to start transcription. Cotranscriptional Processing of RNA State of phosphorylation of CTD determines the type of proteins that can associate with the CTD (thus defining cotranscriptional process). 5’ end of pre-mRNA is capped with 7-methylguanosine. This protects the transcript from degradation; capping is also necessary for translation of mature mRNA. Cotranscriptional Processing 3’ end of the transcript typically contains AAUAAA or AUUAAA. This sequence is recognized by an enzyme that cleaves the newly synthesized transcript ~20 nucleotides downstream. At the 3’ end, a poly(A) tail consisting of 150 200 adenine nucleotides is added. Polyadenylation is another characteristic of transcription in eukaryotes. Complex Patterns of Eukaryotic RNA Splicing Different mRNA can be produced; different a-tropomyosin can be produced. Alternative splicing is a mechanism for gene regulation. Gene product can be different in different cell types and at different stages of development. Intron Splicing: Conserved Sequences exons - coding sequences introns - noncoding sequences Small nuclear ribonucleoprotein particles (snRNPs) recognize consensus splice junction sequence of GU/AG. snRNPs are complexes of protein and small nuclear RNA (snRNA). Several snRNPs comprise a spliceosome. Spliceosome directs the removal of introns and joining of exons. Spliceosome Assembly and Function Spliceosome interacts with CTD and attaches to pre-mRNA. snRNAs in spliceosomes direct alignment of the splice sites. One end of conserved sequence attaches to conserved adenine in the intron. The “lariat” is released and adjacent exons are joined. Reactions in Exon Splicing Self-Splicing Reaction RNA molecules can act somewhat like enzymes (ribozymes). In the protozoan Tetrahymena, the primary transcript of an rRNA can excise a 413-nucleotide intron from itself. These self-splicing introns are an example of RNA that can catalyze a reaction. Transfer of Information in Prokaryotes and Eukaryotes What are the similarities? What are the differences? Colinearity of Gene and Protein genotype DNA DNA sequence transcription RNA translation protein function phenotype organism amino acid sequence Colinearity of Gene and Protein “The linear sequence of nucleotides in a gene determines the linear sequence of amino acids in a protein.” Mutant alleles of trpA gene differed in the position of the mutation at the DNA level, which corresponded to position of amino acid substitution in the gene product. Colinearity of mutations and altered amino acids in a subunit of tryptophan synthetase from E. coli C. Yanofsky, 1967. Scientific American Tryptophan Synthetase Activity indole-3-glycerol phosphate a subunit glyceraldehyde 3-phosphate indole serine b subunit tryptophan Molecular Basis for Relationship between Genotype and Phenotype genotype DNA DNA sequence transcription RNA translation protein function phenotype organism amino acid sequence