1. Chapter 17 PROTEIN SYNTHESIS: FROM GENE TO PROTEIN

2. Central Dogma of Biology Flow of genetic information:

3. RNA (ribonucleic acid) DIFFERENCES DNARNA deoxyribose sugar double strand bases A,T,C,G found in: nucleus, mitochondria, chloroplasts ribose sugar single strand bases A,U,C,G found in: nucleus, cytosol, ribososomes many functions

4. RNA has many functions in the cell. 1. pre-mRNA: precursor to mRNA, newly transcribed and not edited 2. mRNA: carries the code from DNA that specifies amino acids 3. tRNA: carries a specific amino acid (anticodon)to ribosome 4. rRNA: makes up 60% of the ribosome; site of protein synthesis 5. snRNA: small nuclear RNA; part of a spliceosome (in RNA splicing) 6. srpRNA: a signal recognition particle that binds to signal peptides 7. RNAi: interference RNA; a regulatory molecule 8. ribozyme: RNA molecule that functions as an enzyme

5. 3 types RNA directly involved in making proteins 1. messenger RNA (mRNA) single uncoiled long strand - transmits DNA info during protein synthesis - serves as template to assemble amino acids 2.transfer RNA (t RNA) - carries amino acids to ribosome 3.ribosomal RNA (r RNA) makes up large part (2/3) of ribosome - globular

6. PROTEIN SYNTHESIS/GENE EXPRESSION Formation of proteins using information coded on DNA and carried out by RNA. one gene = one RNA molecule Gene: region of DNA whose final product is either a polypeptide or RNA molecule A gene is expressed when protein synthesis is occurring.

7. The Genetic Code How is information necessary for creating proteins encoded in the RNA? The genetic code from DNA is transcribed onto m RNA by Codons. For each gene, one DNA strand is the template strand mRNA (5’  3’) complementary to template mRNA triplets (codons) code for amino acids in polypeptide chain

8. Code word/Codon (triplet): specific group of 3 successive bases on DNA and mRNA - codes for a specific amino acid to be placed on the protein chain - 20 biological amino acids, but more than 20 codons Like “genetic words” DNA code words: ACT, GCA, TTA RNA codons: UGA, CGU, AAU

9. How many combinations of code words can we make from 4 bases? 64 different codon combinations ( 4 3 = 64) ** each code word always codes for same amino acid** Redundancy: 1+ codons code for each of 20 AAs Reading frame: groups of 3 must be read in correct groupings This code is universal: all life forms use the same code

10. Ala: Alanine Cys: Cysteine Asp: Aspartic acid Glu: Glutamic acid Phe: Phenylalanine Gly: GlycineHis: Histidine Ile: Isoleucine Lys: LysineLeu: Leucine Met: MethionineAsn: Asparagine Pro: ProlineGln: GlutamineArg: ArginineSer: Serine Thr: ThreonineVal: ValineTrp: TryptophaneTyr: Tyrosisne

11. It is possible for non-Watson-Crick base pairing to occur at the third codon position. This has phenomenon been termed the wobble hypothesis.

12. How do these code words affect protein synthesis? Order of code words codes for Order of amino acids codes for Specific type of protein

13. BUILDING OF PROTEINS - DNA unzips - original strand of DNA acts as template for m RNA (only one strand of DNA molecule needed for transcription) euchromatin: uncoiled areas of DNA, active site of transcription antisense /non-template strand: non coding strand of DNA sense/template strand: coding strand of DNA

14. 2 Stages of Protein Synthesis

15. Stages of Protein Synthesis I.Transcription (nucleus) Process where mRNA is produced from DNA Transcription unit: stretch of DNA that codes for a polypeptide or RNA

16. Transcription 1.Initiation (prokaryotes) RNA polymerase binds directly to promoter in DNA - promoter : region of DNA that initiates transcription of a particular gene. - located near the transcription start sites of genes on sense strand - located upstream on the DNA (towards the 5’ region of the antisense strand) **can be about 100–1000 base pairs long** nontemplate strand template strand

17. Steps of transcription 1. Initiation (eukaryotes) A. The promoter site on the DNA contains a sequence called a TATA box - TATAAAA - recognized by RNA polymerase ll - can be up to 25 bases upstream from point of transcription B. Transcription factor proteins and RNA polymerase ll bind to promoter section of DNA and unwinds the part of the DNA to be transcribed. (this is the structural gene: codes for a single protein)

18. 2. Elongation A. RNA polymerase reads DNA template strand B. Complementary nucleotides are added to the 3' end of RNA using information in DNA as instructions **Polymerases always work from the 3' to the 5' end of the coding strand of DNA (template); thus the antiparallel structure it is forming is going from the 5' to 3’ direction. C. As RNA polymerase moves, it untwists DNA, then rewinds it after mRNA is made D. Once RNA nucleotides are attached to DNA chain, codons are in proper order

19. 3.Termination A.RNA polymerase transcribes a terminator sequence in DNA B.mRNA and polymerase detach. * prokaryotes- mRNA ready to use * eukaryotes- pre-mRNA, will undergo further modifications transcription animation

20. RNA Processing Occurs After Transcription 1. Alteration of pre-mRNA ends - 5’ cap (modified guanine) and 3’poly-A tail (50-520 A’s) are added - functions: help export mature mRNA from nucleus protect mRNA from enzyme degradation help ribosomes attach to mRNA

21. RNA Processing Occurs After Transcription 2.RNA Splicing - Pre-mRNA has introns (noncoding sequences) and exons (codes for amino acids) - Splicing- introns cut out, exons joined together - Once RNA is spliced it can move out of the nucleus to be translated

22. RNA Splicing, cont. snRNPs small nuclear ribonucleoproteins snRNP = snRNA + protein Pronounced “snurps” Recognize splice sites snRNPs join with other proteins to form a spliceosome Spliceosomes catalyze the process of removing introns and joining exons Ribozyme = RNA acts as enzyme RNA splicing animation

23. Importance of Introns Functions not known for most introns Some regulate gene expression Alternative RNA Splicing: produce different combinations of exons Depends which segments are treated as exons during splicing One gene can make more than one polypeptide 20,000 genes  100,000 polypeptides Changes in gene expression may confer an evolutionary advantage

24. Prokaryotic vs Eukaryotic Gene Expression Prokaryotes Transcription and translation both in cytoplasm RNA poly binds directly to promoter No introns No RNA splicing Eukaryotes Transcription in nucleus; translation in cytoplasm DNA in nucleus, RNA travels in/out nucleus RNA poly binds to TATA box & transcription factors RNA contains introns RNA splicing

25. Translation (in cytoplasm at ribosome) - process whereby protein is synthesized from mRNA - newly synthesized mRNA moves from nucleus to ribosome in cytoplasm - gene has 3x more nucleotides than the protein it makes Ex: 100 a.a. = 300 nucleotides - components of translation mRNA- message tRNA- interpreter ribosome- site of translation

26. Transfer RNA (t RNA) - function: transfers amino acids to ribosome - 20 types – one for each amino acid (specific) - structure (cloverleaf) - found in cytosol Aminoacyl-tRNA-synthetase: enzyme that binds tRNA to specific amino acid

27. Ribosomes - made in nucleolus -2 subunits make up ribosome - about 2/3 is r-RNA and 1/3 is protein - smaller subunit has binding site for mRNA - normally apart in cytoplasm, come together during protein synthesis

28. Steps of translation 1. Initiation A. 5’ end of m RNA binds to small subunit B. initiator tRNA carrying Met attaches to P site C. large subunit attaches (ribosome ready for protein synthesis) - sites: locations on ribosome where tRNA anticodons attach P (peptidyl) site- holds aa chain A (aminoacyl) site- holds aa to be added E (exit) site- exit for tRNA * start codon (AUG) will be at the site on mRNA where this occurs ** anticodon on first tRNA will always be UAC, amino acid 1 will always be methionine

29. 2. Elongation A. t-RNA with a specific anticodon binds a specific amino acid. This happens for several t-RNAs and proper corresponding amino acids in the cytoplasm. ATP: energy source used to bind the amino acid to the t-RNA. Aminoacyl-tRNA synthase: enzyme that does the binding. B. First tRNA binds to P site, second tRNA binds to A site (anticodons are complementary to mRNA codons) C. Peptidyl transferase reaction occurs: #1 a.a. joins to #2 a.a. D. Ribosome moves down mRNA and first tRNA is released to be used over again - translocation: movement of ribosome down mRNA E. Amino acids continue to be added to protein chain thru same mechanism - peptidyl synthase: enzyme that joins a.a. together

30. 3.Termination A. stop codon is reached (UAA, UGA, or UAG). B. release factor binds to stop codon and polypeptide is released C. subunits dissociate (can be used over again) D. protein is released into cell E. mRNA is broken down by cell (not be used again – only once) F. tRNA is released into cell (used over again)

31. Speed of Translation - process occurs from minutes to hours in an organism - Polyribosomes: strings of ribosomes that can translate many copies of a polypeptide very quickly - During synthesis, polypeptide chain coils and folds spontaneously Protein synthesis animation Protein synthesis animation 2

32. Where to proteins go once synthesized? Free ribosomes (floating in cytosol) make proteins that stay in cytosol to perform functions Bound ribosomes (attached to ER) make proteins for secretion Use signal peptide to target their location make proteins of the endomembrane system nuclear envelope, ER, Golgi, lysosomes, vacuoles, plasma membrane)

33. Signal Mechanism Signal peptide: 20 AA at leading end of polypeptide determines destination Signal-recognition particle (SRP): brings ribosome to ER

34. Mutations Changes in genetic code of a cell Source of new genes and diversity of genes among organisms Mutagen: substance that causes mutations Radiation, chemicals, viruses 1.Large scale (chromosome) Involve large segments of chromosome Cause disorders or death Types: duplications, large deletions, translocation, inversion, non-disjunction 2.Small scale Single nucleotide-pair substitutions Nucleotide-pair insertions or deletions (one or more pairs)

35. Large Scale Chromosome Mutations Review

36. Small Scale Mutations Point mutations: alter 1 base pair of a gene 1. Base-pair substitutions – replace 1 with another Silent: no change in amino acid due to redundancy Missense: change one amino acid into another Nonsense: change into stop codon, results in shorter non-functional polypeptide 2.Frameshift: alters reading frame of RNA causes non-functional proteins Insertions: addition of nucleotide/s Deletions: removal of nucleotide/s

37. Base Pair Substitutions Substitution: Silent (no effect)

38. Base Pair Substitutions Substitution- Missense (change of one amino acid to another)

39. Sickle Cell Anemia: point mutation

40. Base Pair Substitutions Substitution: Nonsense (change into stop codon)

41. Frameshift Insertion: shifts frame to right

42. Frameshift Deletion: shifts frame to left, premature termination

43. Summary of Protein Synthesis