Gene Function

1. Gene Function 19 Jan, 2005

3. Transfer of information • DNA  RNA  polypeptide • Complementary base pairing transfers information • during transcription to form RNA • during translation between codon and anticodon • DNA binding proteins • recognize double- or single-stranded DNA • recognize specific nucleotide sequences • are coded by genes • have variety of important functions

4. RNA • Transcription: copying nucleotide sequence of DNA into RNA • forms RNA transcript • DNA may be transcribed multiple times • RNA • single-stranded polynucleotide • contains ribose sugar • contains the pyrimidine uracil (U) • hydrogen bonds with A • 5’ and 3’ ends critically important

5. RNA Nucleotides

6. Transcription

8. 5’UTR 3’UTR coding region Transcription steps • Initiation • at 5’ end of gene • binding of RNA polymerase to promoter • unwinding of DNA • Elongation • addition of nucleotides to 3’ end • rules of base pairing • requires Mg2+ • energy from NTP substrates • Termination • at 3’ end of gene • terminator loop (prokaryote) or processing enzyme

9. Promoters

11. Eukaryote RNA processing • 5’ end: capping • addition of 7-methylguanosine • linked by three phosphates • 3’ end: poly(A) tail • addition of up to 200 adenine nucleotides • downstream of AAUAAA polyadenylation signal • Intron removal by spliceosome • all introns have 5’GU and 3’AG recognition sequence (GU – AG rule) • snRNPs of spliceosome provide catalysis • intron excised as lariat, destroyed Some nonprotein- encoding genes have self-splicing introns.

15. Processing Overview

16. Protein structure • Protein is polymer of amino acids (polypeptide) • each amino acid has R group conferring unique properties • amino acids connected by peptide bond • each polypeptide has amino end and carboxyl end • Structures • primary: amino acid sequence • secondary: hydrogen bonding, -helix and -sheet • tertiary: folding of secondary structure • quaternary: two or more tertiary structures • Shape and function determined by primary structure encoded by gene

20. Translation • mRNA is translated by tRNA at ribosome • nucleotide sequence is read three nucleotides at a time • each triplet is called a codon • each amino acid has one or more codons • 64 possible codons (4  4  4) = genetic code • used by all organisms with few exceptions • Genetic code specifies 20 different amino acids (sometimes selenocysteine)

21. Codon translation • tRNA • anticodon consists of 3 nucleotides • base pairs with codon in antiparallel fashion • 3’ acceptor end attaches amino acid • attachment catalyzed by aminoacyl-tRNA synthetases • one for each different tRNA • Wobble hypothesis • permits third nucleotide of anticodon (5’ end) to hydrogen bond with alternative nucleotide • permits a tRNA to translate more than one codon

25. Translation at the ribosome • Ribosome • large subunit • small subunit • 3 ribosomal sites • A site (amino site), accepts incoming charged tRNA • P site (polypeptide site), peptide bond • E site (exit site), tRNA exits ribosome • Amino terminus synthesized first, beginning near 5’ end of mRNA

28. Protein function • Function determined by amino acid sequence • Colinearity between DNA nucleotide sequence and amino acid sequence of protein • Two broad types of protein • structural proteins • active proteins, including enzymes • Proteins often have specialized domains

30. Malfunctioning alleles • Mutation alters gene function by altering structure/function in product • wild-type: normal allele • designated by plus (+) sign • example: arg-3+ • mutation: change in nucleotide sequence • sometimes designated by minus (–) sign • Nutritional mutants • prototroph: wild-type, synthesizes nutrients • auxotroph: mutant, fails to make essential nutrient (e.g., amino acid)

32. Types of mutation • Mutant site: area of nucleotide change • Three types of mutation • substitution of different amino acid • e.g., 5’GGA3’  5’GAA3’, gly  glu • premature stop codon • e.g, 5’GGA3’  5’UGA3’, gly  stop • frameshift • insertion or deletion of one or two nucleotides alters reading frame from point of change • all downstream codons altered

33. Effect of mutation • Often reduces or eliminates protein function • leaky mutation: reduced function • null mutation: no function • silent mutation: no change in function, though amino acid sequence may be changed • Mutations in information transfer • mutations in exon-intron junction • mutations in promoter or regulatory sequences • mutations in UTRs

34. Dominance and recessiveness • Recessive genes typically produce little or no product. One dose of wild-type gene produces sufficient product, resulting in dominant phenotype (haplo-sufficiency) • Nomenclature • recessive genes, lower case italicized letter, e.g., a • dominant gene, upper case italicized letter, e.g., A • Genotypes • A/A, (a+/a+) homozygous dominant • A/a, (a+/a) heterozygous • a/a, homozygous recessive normal phenotype

36. Haplo-insufficiency • Wild-type gene provides insufficient product to fulfill normal cell function • in this case, defective gene is dominant • B+/B+, homozygous recessive • B/B+, heterozygous • B/B, homozygous dominant defective phenotype

39. Assignment: Concept map, solved problems 1 and 2, basic problems 2, 8 through 12, challenging problems 18, 21, 23-25 Continue with web-based NCBI tutorial sections from Introduction to Using BLAST to compare sequences.