Chapter 12 From DNA to Protein: Genotype to Phenotype

1. Chapter 12 From DNA to Protein: Genotype to Phenotype Biology 101 Tri-County Technical College Pendleton, SC

2. From past to present… • One gene = one protein • One gene = one enzyme • Beadle and Tatum • Altered phenotype = altered enzyme protein • One gene = one polypeptide • **Function of one gene is to control (direct) the synthesis (production) of ONE polypeptide

3. Comparing RNA to DNA • RNA usually consists of only ONE polynucleotide strand • Chargaff’s rule NOT applicable • Pentose is ribose • AUGC • Always constructed as single strand but may fold over and base pair with itself

4. Central Dogma • States DNA codes for production of RNA, RNA codes for production of protein, and protein does NOT code for production of protein, RNA, or DNA • Took a long time to get to here…remember Hershey and Chase’s bacteriophage experiments

5. Central Dogma Visual

6. Making Stuff…so to speak • RNA molecule forms as complementary copy of one DNA strand of a particular gene in process called transcription • Forms messenger RNA (mRNA) which travels to cytoplasm where it serves as template for protein synthesis • Adapter molecule (Crick) that can bind a specific AA and recognize sequence of nucleotides with another region is called transfer RNA (tRNA) • Sequence of 3 bases called ANTICODON

7. Stuff, cont. • tRNA adapter line up on mRNA so that AA are in proper sequence for growing polypeptide chain in process called translation • Translation requires third type of RNA called ribosomal RNA (rRNA) which is RNA complexed with proteins • Given gene (sense) transcribed into mRNA; tRNAs translate sequence of bases in mRNA into appropriate sequence of AAs in assocation with ribosomes (rRNA and protein)

8. Making Stuff Visual

9. 3 Steps of Transcription • Initiation, elongation, and termination • Begins at promoter (sequence of DNA to which RNA polymerase binds • At least one promoter for each gene • Part of promoter is initiation site where transcription begins • RNA polymerase moves in 3’ to 5’ direction • Which means that transcription proceeds in 5’ to 3’ direction ONLY

10. Steps, cont. • RNA polymerase unwinds DNA about 20 bases at a time • RNA polymerase then adds RNA nucleotides to the 3’ end of growing strand of RNA • Termination site reached, RNA polymerase detaches from DNA • Unlike DNA polymerases, RNA polymerases DO NOT inspect their work

11. Steps, cont. • Transcription errors = one mistake for every 103 to 105 bases • Chalk talk time on transcription • One can bet the farm an opportunity will be provided to demonstrate understanding of this process

12. Understanding the codon • Codon is sequence of 3 bases on mRNA that “code” (specify) a particular AA or a stop • The genetic code will be provided • AUG (methionine) is universal start codon • UAA, UAG, and UGA are stop codons • MUST know how to read/apply the code • Care to guess how they figured out how many bases must be in a codon?

13. I like the term degenerate… • There are 64 different 3 letter codons • 61 of them call for a specific AA • An AA may be represented by more than one codon, so we now say code is redundant • Unambiguous means that specific codon requires a specific AA • It is NOT roll your own, or any ole AA will do • More than one way to say “put valine here” but NOT any ole AA here

14. Putting it together • Given a sequence bases in DNA (either sense or nonsense) build the Doc everything else. • One can bet the farm this will show up again (and again) • Hot damn, it be chalk talk time again

15. Ribosome structure and function • Each ribosome consists of 2 subunits: small and large • Different proteins and RNA’s in subunit held together by ionic and hydrophobic forces • Ribosome can combine with any mRNA and all tRNAs and can be used to make different polypeptide products

16. Ribosomes, cont. • Ribosome simply molecular factory where proteins are assembled • Large subunit contains 4 sites • T (transfer) site is where tRNA carrying an amino acid first lands on ribosome • A (amino acid) site is where tRNA anticodon binds to mRNA codon, lining up correct AA to be added to growing chain

17. Ribosomes, cont. • P (polypeptide) site is where tRNA adds its amino acid to growing chain • E (exit) site is where tRNA, having given up its AA resides before leaving ribosome • **Codon-anticodon interactions and peptide bond formation occur ONLY at A and P sites • Polysome: more than one ribosome translating a strand of mRNA • 70S and 80S ribosomes and endosymbiotic theory of eukaryotic evolution

18. Take three steps and translate • Initiation, elongation, and termination • Initiation depends on initiation factors (proteins) which direct process and use GTP • Energy for elongation also comes from GTP • Termination depends on “stop” codon • Stop codon does NOT bind any tRNA but does bind release factor which causes water molecule to be added to PP chain instead of AA • Causes separation of complete protein from ribosome • Hot damn…chalk talk time on translation

19. Protein Destination • All protein synthesis begins on free ribosomes in cytoplasm • As PP chain made, into in its AA sequence gives it one of two sets of directions • Finish translation and be released into cytoplasm • Stop translation and go to rough ER and finish there • Can be sent anywhere in cell, or lacking specific instructions, it is secreted from cell • After translation, some PPs have short exposed sequence of AAs that act like postal “zip code”

20. Destination, cont. • Signal sequences are at N terminus or in interior of AA chain • Have conformation that allow binding to specific receptor proteins (docking proteins) on outer membrane of appropriate organelle • Receptor forms channel in membrane • If specific hydrophobic sequence of ~25 AAs occurs at beginning of PP chain, finished product destined for ER, lysosomes, PM, or secretion from cell

21. Destinations, cont. • Sugars can be added as part of address • Addressing of protein to destination is property of its AA sequence and so is genetically determined • Mucoplidosis II (I-cell disease) caused by lack of essential enzyme for formation of lysosomal targeting signal

22. Destinations, cont. • Proteins destined for lysosome never get there but stay in Golgi apparatus where they form I (inclusion) bodies or are secreted from cell • Inability to perform normal lysosome functions leads to progressive illness and death in childhood

23. Protein Modification • **Review of precursor concept • Most proteins modified both covalently and noncovalently • Essential for final functioning of protein • Proteolysis is cutting of PP chain • Some proteins made from polyproteins cut into final products by enzymes called proteases

24. Modification, cont. • Glycosylation is adding of sugar to protein • Important in addressing; 3-dimensional structure; recognition of proteins at cell surface; and in stabilizing stored proteins • Phosphorylation is addition of phosphate group to protein • Change 3-dimensional structure (often exposing active site or binding site for another protein

25. Making sense of nonsense… • Mutations are heritable changes in genetic information • True, but…sudden, permanent change in DNA • Point mutations = mutations of single genes • One allele becomes another because of small alterations in sequence or number of nucleotides

26. Making sense, cont. • Point mutation result from addition or subtraction of a base or the substitution of one base for another in DNA (exon/intron) • Genetic code is redundant so some point mutations result in NO change in AA sequence and are called silent/synonymous • Some base substitution mutations change message so one AA substituted for another and are called missense

27. Making sense, cont. • Sickle-cell disease results from ONE wrong AA • Missense may cause protein NOT to function but usually just reduce efficiency • Nonsense mutation cause a “STOP” codon to be inserted in mRNA to be translated • Results in nonfunctional product • Frame shift mutation occurs when base pairs are inserted into or deleted from DNA • Throw message out of synch • Shift the reading frame-bad news, big time

28. Some Key Terms • One more time…with feeling…yehaw!! • Chromosomal mutations change position or direction of DNA segment without removing any genetic info or may cause segment to be irretrievably lost • Let’s talk karyotype • **Review: deletion, duplication, inversion, and translocation