Storage and use of genetic information

1. Storage and use of genetic information • The genetic code • Three bases (in a row) specify an amino acid • Transcription • The synthesis of a mRNA, complementary to one of the DNA strands, containing the genetic code • Translation • Proteins and rRNAs in the ribosome along with tRNAs translate the genetic code into proteins. • Post-translational modification • Proteins are altered after synthesis

2. The Genetic Code • Four bases taken how many at a time? Need to code for 20 different amino acids. • Each base = 1 amino acid: only 4 • Every 2 bases = 1 a.a.: 16 combinations, 4 short. • Every 3 bases: 64 combinations, enough. • Every 3 bases of RNA nucleotides: codon • Each codon is complementary to 3 bases in one strand of DNA • Each codon (except for T →U switch) is the same as 3 bases in the other DNA strand.

3. More about the Genetic Code • The code is • Unambiguous: each codon specifies 1 amino acid • Degenerate: a particular amino acid can be coded for by several different codons. • Ordered: similar codons specify the same amino acid. • Commaless, spaceless, and non-overlapping : each 3 bases is read one after the other. • Punctuated: certain codons specify “start” and “stop”. • Universal: by viruses, both prokaryotic domains, and eukaryotes (except for some protozoa, mitochondria).

4. The Genetic Code-2 http://www.biology.arizona.edu/molecular_bio/problem_sets/nucleic_acids/graphics/gencode.gif

5. Wobble • Crick’s Wobble Hypothesis • The code is “ordered” • The first 2 positions are more important • When lining up with the anticodon of the tRNA, the third position doesn’t bind as tightly, thus a looser match is possible. • Because of this flexibility, a cell doesn’t need 61 different tRNAs (one for each codon). • Bacteria have 30-40 different tRNAs • Plants, animals have up to 50.

6. Colinearity • Archibald Garrod (1908) determined that genes must code for enzymes by studying metabolic diseases (black urine). But what relationship? • Beadle and Tatum (1941): one gene one enzyme, there is a one to one correspondence between DNA and protein • Mutation in DNA changes nucleotide sequence, changes amino acid sequence in protein. • Not completely true in eukaryotes because of introns

7. The code is colinear with protein sequence • Bacteriophage MS2 • An RNA virus with 3 genes, including a viral coat protein. • Amino acid sequence of coat protein determined, 1970 • Sequence of coat protein gene determined, 1972 • Correspondence between codons and amino acids exactly as predicted.

8. Ribosome structure Large and small subunits. Eukaryotic: 60S & 40S = 80S Prokaryotic: 50S & 30S = 70 S Large subunit: 2 -3 rRNAs and many proteins Small subunit: 1 rRNA and many proteins http://www.emc.maricopa.edu/faculty/farabee/BIOBK/ribosome.gif

9. Size in Svedbergs • S is a unit based on ultracentrifugation • What affects how fast particles move toward the bottom of the tube? • Mass, density, and shape (which affects friction) • Example: supercoiled DNA moves faster than relaxed DNA. • Because of these factors, S units are not additive • 50S + 30S prokaryotic subunits make a 70S ribosome.

10. Components of ribosomes • Multiple copies exist of ribosomal genes • Def. of “gene” extended to DNA that codes for rRNA • Multiple copies means moderately repetitive DNA • Cells can crank out lots or rRNA (and r-proteins) • Transcription results in RNA requiring processing • Pre-RNA cut into rRNAs (and tRNAs) • rRNA NOT just structural: are ribozymes, carry out the actual protein synthesis.

11. About tRNAs-1 • Coded for by tRNA genes • Post-transcriptional modification • tRNAs have some bases changed • tRNAs interact with rRNA during protein synthesis • tRNAs must have amino acid attached • Enzymes: aminoacyl tRNA synthetases • 20 different enzymes, one for each aa. • Enzymes recognize shape of tRNA

12. About tRNAs-2 • tRNA structure must be highly conserved • Must be folded so that both the synthetases recognize it, and it fits properly on the ribosome. • Errors in translation result in bad proteins; mutations in tRNA genes are selected against.

13. tRNA 3D structure: The familiar loops of the 2D structure are labeled. 3’ end: Attaches to amino acid. Decoder end: Complementary to codon. hto-b.usc.edu/~cbmp/2001/ tRNA/trna%20s1.jpg

14. Translation • mRNA: provides message to be translated. • Ribosomes: functional workbench for synthesis. • tRNA: bring aa to ribosome, decode mRNA. • Aminoacyl tRNA synthetases: enzymes that attach amino acids to tRNAs. • Protein factors: help move process along: initiation, elongation, and termination. • Process is similar, but different between prokaryotes and eukaryotes.

15. Initiation and Termination of protein synthesis • AUG is always the first codon (initiator codon) • Establishes an “open reading frame” (ORF) • Ribosome begins synthesis with a methionine • In bacteria, it is N-formylmethionine (fMet) • After synthesis , either formyl group is removed or entire fMet is removed (Met in eukaryotes) • Three codons serve as termination codons: • UGA, UAG, UAA; any one can be a stop signal • Do NOT code for an amino acid • Cause translation to end; protein is completed formyl

16. Translation-1 • Initiation • Small subunit, mRNA, met-tRNA, IFs, GTP • mRNA: sequence for binding to ribosome needed • prokaryotes: Shine-Delgarno • Eukaryotes: Cap and Kozak sequence • (GCC)RCCATGG where R is a purine • First tRNA is fMet-tRNA in prokaryotes • IFs are protein Initiation Factors • GTP needed for energy • When all have come together, Large subunit added

17. Translation-2 • Ribosome has 3 sites • AA site where tRNA-aa first sits in • P site where tRNA with growing peptide sits • E or Exit, site transiently occupied by used tRNA • Elongation, with help of EFs and GTP • tRNA with new aa sits in A site • Stays in A site if anticodon on tRNA is complementary to codon on mRNA. • tRNA in P site transfers growing chain to new aa • Catalyzed by rRNA • Ribosome moves relative to mRNA and tRNAs • tRNAs now in new sites, new codon lined up

18. Ribosome schematic http://staff.jccc.net/pdecell/proteinsynthesis/translation/elongation12.gif

19. Translation-3 • Termination • When stop codon is in A site, no tRNA binds • GTP-dependent release factor (protein) removes polypeptide from tRNA in P site. All done. • Ribosomal subunits typically dissociate. • Do a Google Search for translation animation • Many hits. Note presence, absence of E site • Note shape of ribosomes • Note whether role of rRNA in catalysis is shown

20. Nonsense mutations and suppressors • A mutation may change a normal codon to a stop codon; protein synthesis ends prematurely. (nonsense mutation) • A second mutation can cure the original: a “suppressor”. • If the gene for a tRNA is mutated in the anticodon so that the stop codon is now read by the tRNA.

21. Polysomes and Polycistronic mRNA • In eukaryotes, when mRNA enters the cytoplasm, many ribosomes attach to begin translation. A mRNA w/ many ribosomes attached = polysome. • In eukaryotes, the mRNA for a single gene is processed and translated; in prokaryotes, mRNA can be polycistronic, meaning several genes are on the same mRNA and are translated together • With no nucleus, translation can begin in prokaryotes before transcription is over.

22. Polysomes Multiple ribosomes attach to the mRNA and begin translating. Strings of ribosomes can be seen attached to the mRNA. http://opbs.okstate.edu/~petracek/Chapter%2027%20Figures/Fig%2027-29b-bottom.GIF www.cu.lu/labext/rcms/ cppe/traducti/tpoly.html

23. Eukaryotic, prokaryotic differences • mRNA lifetime is different • Cap, tail, introns in eukaryotes • Shine-Delgarno vs. Kozak sequence & cap • Size of ribosomes: 70S vs. 80S • fMet-tRNA vs. Met-tRNA • Eukaryotic: attachment of ribosomes to ER • Polypeptides extruded through tunnel in large subunit, directly into ER

24. Proteins vs. polypeptide • Common usage: • Polypeptide is a string of amino acids • Doesn’t imply function • Doesn’t imply 3D shape • Protein implies functionality and 3D shape • Thus a “protein” can have a quaternary structure and be made of several different polypeptides.

25. Review of protein structure String of amino acids, covalently attached by peptide bonds; directional (N terminus, C terminus).

26. Primary structure The particular amino acids and the order that they are in. 20 different amino acids connected by peptide bonds; 100-1000 amino acids in a peptide chain.

27. Secondary structure Amino acid chain twists in space, held in place by hydrogen bonds. Forms alpha helix or beta pleated sheet.

28. Tertiary structure 3-D folding of the protein chain in space; the shape is determined from the primary structure and from the secondary structure (which itself depends on the primary structure. The primary structure depends on the info encoded in the DNA Protein structure slides from

29. Quaternary structure Individual polypeptide chains aggregate to form a single functional unit. Individual polypeptides (protein subunits) may be switched out to give the multipart protein a different function or specificity. Many proteins that act on DNA or RNA have a quaternary structure.

30. Domains Different exons actually code for parts of proteins that fold into discrete areas. May be involved in evolution of proteins and their functions.