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Gene Expression and Protein Synthesis

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

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Gene Expression and Protein Synthesis

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  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

  14. 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

  15. 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

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

  17. The Genetic Code

  18. 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

  19. 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

  20. 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

  21. 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

  22. Fig. 24.16

  23. The Genetic Code

  24. 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

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

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

  27. 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

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

  29. Polypeptide Elongation

  30. 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

  31. Elongation

  32. 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

  33. 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)

  34. 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

  35. 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

  36. 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

  37. 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)

  38. 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

  39. 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

  40. 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

  41. Recombinant DNA

  42. Recombinant DNA

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