Gene Expression and Protein Synthesis

1. Gene Expression and Protein Synthesis Bettelheim, Brown, Campbell and Farrell Chapter 26

2. Introduction • The central dogma of molecular biology • information contained in DNA molecules is expressed in the structure of proteins • gene expression is the turning on or activation of a gene

3. Transcription • Transcription: the process by which RNA polymerase synthesizes RNA from a DNA template • DNA double helix begins to unwind near the gene to be transcribed (helicase) • Only one strand of the DNA is transcribed • (Different from DNA replication) • Ribonucleotides assemble along the unwound DNA strand in a complementary sequence • Enzymes called RNA polymerases(poly) catalyze transcription: • poly I for rRNA, poly II for mRNA, poly III for tRNA

4. Transcription

5. Transcription Overview 1.Unwinding of DNA (Helicase) 2.Binding of Enzyme • Transcriptional factors • Promoter, TATA boxes • Initiation sequence 3.Elongation (Synthesis of RNA strand) • Stopped at termination sequence 4.Post-Transcriptional Processing • Capping, methylation, poly A addition, splicing • Think about this as changing it into its “working” form

6. Transcription • A eukaryotic gene has two parts • structural gene that is transcribed into RNA • made of exons and introns • regulatory gene that controls transcription • not transcribed itself but has control elements, such as the promoter • promoter is unique to each gene

7. Transcription • sequence of bases on the DNA strand called an initiation signal—tells enzyme where to start • promoters also contain consensus sequences such as the TATA box in which the two nucleotides T and A are repeated many times • a TATA box lies approximately 25 base pairs upstream of initiation signal

8. Transcription • all three RNA polymerases interact with their promoter regions via transcription factors that are binding proteins • after initiation, RNA polymerase zips up the complementary bases in a process called elongation • Elongation (synthesis) of new strand is in the 5’ -> 3’ direction • Termination sequence at end of gene (STOP)

9. Processing of RNA transcript • Ribozymes cut out introns and splice the exon ends together to form m-RNA

10. Fig. 25.2, p.622

11. Transcription • The RNA transcripts made functional by post-transcription modification • transcribed mRNA is capped at both ends • 5’ end acquires a methylated guanine • 3’ end acquires a poly A tail that may contain from 100 to 200 adenine residues • After ends are capped, the introns are spliced out • tRNA undergoes similar modification • capped, methylated, and trimmed • rRNA undergoes methylation

12. DNA-RNA Replication Template DNA strand: New RNA strand: ATTCGTAAAGGTC UAAGCAUU

13. RNA in Translation • mRNA, rRNA, and tRNA all involved in translation • protein synthesis takes place on ribosomes • a ribosome has one large and one small unit • in higher organisms, the larger unit is called a 60S ribosome; the smaller unit is called a 40S ribosome • the 5’ end of the mature mRNA is bonded to the 40S ribosome and this unit then joined to the 60S ribosome • together the 40S and 60S ribosomes form a unit on which mRNA is stretched out

15. triplets of bases on mRNA are called codons • the 20 amino acids are then brought to the mRNA-ribosome complex, each amino acid by its own particular tRNA

16. tRNA • each tRNA is specific for only one amino acid • each cell carries at least 20 enzymes (synthetases) specific for one amino acid • activated amino acid binds to the 3’ terminal -OH group of the tRNA by an ester bond (CCA-OH) • at the opposite end of the tRNA molecule is a codon recognition site • the codon recognition site is a sequence of three bases called an anticodon • this triplet of bases is complementary to the codon triplet on mRNA

17. tRNA • The three-dimensional structure of a tRNA

18. The Genetic Code

19. The Genetic Code • Assignments of triplets is based on several types of experiments • one of these used synthetic mRNA • if mRNA is polyU, polyPhe is formed; the triplet UUU, therefore, must code for Phe • if mRNA is poly ---ACACAC---, poly(Thr-His) is formed; ACA must code for Thr, and CAC for His • By 1967, the genetic code was broken

20. Features of the Code • all 64 codons have been assigned • 61 code for amino acids • 3 (UAA, UAG, and UGA) serve as termination signals • AUG also serves as an initiation signal (also Met) • only Trp and Met have one codon each • more than one triplet can code for the same amino acid; Leu, Ser, and Arg, for example, are each coded for by six triplets (degenerate) • the third base is irrelevant for Leu, Val, Ser, Pro, Thr, Ala, Gly, and Arg

21. Features of the Code • for the 15 amino acids coded for by 2, 3, or 4 triplets, it is only the third letter of the codon that varies. Gly, for example, is coded for by GGA, GGG, GGC, and GGU • the code is almost universal: it the same in viruses, prokaryotes, and eukaryotes; the only exceptions are some codons in mitochondria

22. Start with mRNA sequence AUGCCUACUUUAAAA Break into triplets AUG-CCU-ACU-UUA-AAA “Read” code AUG-CCU-ACU-UUA-AAA Met- Pro- Thr- Leu- Lys

23. Fig. 24.16

24. The Genetic Code

25. Translation • Translation: the process whereby a base sequence of mRNA is used to create a protein • There are four major stages in protein synthesis • activation • initiation • elongation • termination

26. Protein Synthesis • Molecular components of reactions at four stages of protein synthesis

27. Amino Acid Activation • Requires • amino acids • tRNAs • aminoacyl-tRNA synthetases • ATP, Mg2+ • Formation on an aminoacyl-tRNA

28. Amino Acid Activation • This two-stage reaction allows selectivity at two levels • the amino acid: the aminoacyl-AMP remains bound to the enzyme and binding of the correct amino acid is verified by an editing site on the tRNA synthetase • tRNA: there are specific binding sites on tRNAs that are recognized by aminoacyl-tRNA synthetases

29. Polypeptide Chain Initiation • In eukaryotes consists of three stages • forming the pre-initiation complex • migration of mRNA • forming the full ribosomal complex

30. Polypeptide Elongation

31. Peptidyl transferase attaches last aa in P site to aa on A site • Whole ribosome moves along one codon on mRNA. • Polypeptide is translocated from A to P site • Empty tRNA leaves • New tRNA comes into A site

32. Elongation

33. Termination • Chain termination requires • termination codons (UAA, UAG, or UGA) of mRNA • releasing factors that cleave the polypeptide chain from the last tRNA and release the tRNA from the ribosome

34. Gene Regulation • Gene regulation: the various methods used by organisms to control which genes will be expressed and when • some regulations operate at the transcriptional level (DNA -> RNA) • others operate at the translational level (mRNA -> protein)

35. Mutations and Mutagens • Mutation: a heritable change in the base sequence of DNA • one copying error for every 1010 bases (average) • mutations can occur during replication • base errors can also occur during transcription in protein synthesis (a nonheritable error) • consider the mRNA codons for Val, which are CAT, CAC, CAG, and CAA • if the original codon is CAT, it may be transcribed onto mRNA as GUC which also codes for Val???? • other errors in replication may lead to a change in protein structure and be very harmful

36. Mutations • Substitution of one base for another • AAUAAUAAU • AAUAAAAAU • Phase shift: Insertion of extra base or deletion of a base so that the triplets are changed and out of phase • AAU AAU AAU Asn-Asn-Asn • AAU AAA UAA U Asn-Lys-Stop

37. Mutations and Mutagens • Mutagen: causes a base change in DNA • Many changes in base sequence caused by radiation and mutagens do not become mutations because cells have repair mechanisms called nucleotide excision repair (NER) • NER can prevent mutations by cutting out damaged areas and resynthesizing them • Not all mutations are harmful • certain ones may be beneficial because they enhance the survival rate of the species

38. Recombinant DNA • Recombinant DNA: DNA from two sources that have been combined into one molecule • Use of recombinant DNA technology has grown tremendously during last 30 years • Technology used to make many products, including “human insulin” and genetically modified organisms (GMO’s)

39. Recombinant DNA Technology • Can use plasmids found in the cells of “weakened strains” of Escherichia coli • plasmid: a small, circular, bacterial DNA • Enzymes called restriction endonucleases cleave DNA at very specific locations • Example: specific for cleavage of the bond between A-G in the sequence • -CTTAAG- -CTTAA G- Red bases are • -GAATTC- -G AATTC- complementarySticky ends

40. Recombinant DNA • Assume “B ” stands for bacterial gene and “H” for human gene • After cutting, double-stranded bacterial DNA now has two “sticky ends”, each with free bases that can pair with a complementary section of DNA • If you cut a human gene with the same restriction endonuclease, you get human DNA with the same sticky ends

41. Recombinant DNA • Human DNA is spliced into the plasmid by the enzyme DNA ligase • Splicing takes place at both ends of the human gene and the plasmid is once again circular • Modified plasmid is then put back into the bacterial cell where it replicates naturally every time the cell divides • These cells now manufacture the human protein, such as human insulin, by transcription and translation

42. Recombinant DNA

43. Recombinant DNA