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Chapter 7

Chapter 7. From DNA to Protein. DNA to Protein. DNA acts as a “manager” in the process of making proteins DNA is the template or starting sequence that is copied into RNA that is then used to make the protein. Central Dogma. One gene – one protein. Central Dogma.

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Chapter 7

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  1. Chapter 7 From DNA to Protein

  2. DNA to Protein • DNA acts as a “manager” in the process of making proteins • DNA is the template or starting sequence that is copied into RNA that is then used to make the protein

  3. Central Dogma • One gene – one protein

  4. Central Dogma • This is the same for bacteria to humans • DNA is the genetic instruction or gene • DNA  RNA is called Transcription • RNA chain is called atranscript • RNA  Protein is called Translation

  5. Expression of Genes • Some genes are transcribed in large quantities because we need large amount of this protein • Some genes are transcribed in small quantities because we need only a small amount of this protein

  6. Transcription • Copy the gene of interest into RNA which is made up of nucleotides linked by phosphodiester bonds – like DNA • RNA differs from DNA • Ribose is the sugar rather than deoxyribose – ribonucleotides • U instead of T; A, G and C the same • Single stranded • Can fold into a variety of shapes that allows RNA to have structural and catalytic functions

  7. RNA Differences

  8. RNA Differences

  9. Transcription • Similarities to DNA replication • Open and unwind a portion of the DNA • 1 strand of the DNA acts as a template • Complementary base-pairing with DNA • Differences • RNA strand does not stay paired with DNA • DNA re-coils and RNA is single stranded • RNA is shorter than DNA • RNA is several 1000 bp or shorter whereas DNA is 250 million bp long

  10. Template to Transcripts • The RNA transcript is identical to the NON-template strand with the exception of the T’s becoming U’s

  11. RNA Polymerase • Catalyzes the formation of the phosphodiester bonds between the nucleotides (sugar to phosphate) • Uncoils the DNA, adds the nucleotide one at a time in the 5’ to 3’ fashion • Uses the energy trapped in the nucleotides themselves to form the new bonds

  12. RNA Elongation • Reads template 3’ to 5’ • Adds nucleotides 5’ to 3’ (5’ phosphate to 3’ hydroxyl) • Synthesis is the same as the leading strand of DNA

  13. RNA Polymerase • RNA is released so we can make many copies of the gene, usually before the first one is done • Can have multiple RNA polymerase molecules on a gene at a time

  14. Differences in DNA and RNA Polymerases • RNA polymerase adds ribonucleotides not deoxynucleotides • RNA polymerase does not have the ability to proofread what they transcribe • RNA polymerase can work without a primer • RNA will have an error 1 in every 10,000 nucleotides (DNA is 1 in 10,000,000 nucleotides)

  15. Types of RNA • messenger RNA (mRNA) – codes for proteins • ribosomal RNA (rRNA) – forms the core of the ribosomes, machinery for making proteins • transfer RNA (tRNA) – carries the amino acid for the growing protein chain

  16. DNA Transcription in Bacteria • RNA polymerase must know where the start of a gene is in order to copy it • RNA polymerase has weak interactions with the DNA unless it encounters a promoter • A promoter is a specific sequence of nucleotides that indicate the start site for RNA synthesis

  17. RNA Synthesis • RNA pol opens the DNA double helix and creates the template • RNA pol moves nt by nt, unwinds the DNA as it goes • Will stop when it encounters a STOP codon, RNA pol leaves, releasing the RNA strand

  18. Sigma () Factor • Part of the bacterial RNA polymerase that helps it recognize the promoter • Released after about 10 nucleotides of RNA are linked together • Rejoins with a released RNA polymerase to look for a new promoter

  19. Start and Stop Sequences

  20. DNA Transcribed • The strand of DNA transcribed is dependent on which strand the promoter is on • Once RNA polymerase is bound to promoter, no option but to transcribe the appropriate DNA strand • Genes may be adjacent to one another or on opposite strands

  21. Eukaryotic Transcription • Transcription occurs in the nucleus in eukaryotes, nucleoid in bacteria • Translation occurs on ribosomes in the cytoplasm • mRNA is transported out of nucleus through the nuclear pores

  22. RNA Processing • Eukaryotic cells process the RNA in the nucleus before it is moved to the cytoplasm for protein synthesis • The RNA that is the direct copy of the DNA is the primary transcript • 2 methods used to process primary transcripts to increase the stability of mRNA being exported to the cytoplasm • RNA capping • Polyadenylation

  23. RNA Processing • RNA capping happens at the 5’ end of the RNA, usually adds a methylgaunosine shortly after RNA polymerase makes the 5’ end of the primary transcript • Polyadenylation modifies the 3’ end of the primary transcript by the addition of a string of A’s

  24. Coding and Non-coding Sequences • In bacteria, the RNA made is translated to a protein • In eukaryotic cells, the primary transcript is made of coding sequences called exons and non-coding sequences called introns • It is the exons that make up the mRNA that gets translated to a protein

  25. RNA Splicing • Responsible for the removal of the introns to create the mRNA • Introns contain sequences that act as cues for their removal • Carried out by small nuclear riboprotein particles (snRNPs)

  26. snRNPs • snRNPs come together and cut out the intron and rejoin the ends of the RNA • Intron is removed as a lariat – loop of RNA like a cowboy rope

  27. Benefits of Splicing • Allows for genetic recombination • Link exons from different genes together to create a new mRNA • Also allows for 1 primary transcript to encode for multiple proteins by rearrangement of the exons

  28. Summary

  29. RNA to Protein • Translation is the process of turning mRNA into protein • Translate from one “language” (mRNA nucleotides) to a second “language” (amino acids) • Genetic code – nucleotide sequence that is translated to amino acids of the protein

  30. Degenerate DNA Code • Nucleotides read 3 at a time meaning that there are 64 combinations for a codon (set of 3 nucleotides) • Only 20 amino acids • More than 1 codon per AA – degenerate code with the exception of Met and Trp (least abundant AAs in proteins)

  31. Reading Frames • Translation can occur in 1 of 3 possible reading frames, dependent on where decoding starts in the mRNA

  32. Transfer RNA Molecules • Translation requires an adaptor molecule that recognizes the codon on mRNA and at a distant site carries the appropriate amino acid • Intra-strand base pairing allows for this characteristic shape • Anticodon is opposite from where the amino acid is attached

  33. Wobble Base Pairing • Due to degenerate code for amino acids some tRNA can recognize several codons because the 3rd spot can wobble or be mismatched • Allows for there only being 31 tRNA for the 61 codons

  34. Attachment of AA to tRNA • Aminoacyl-tRNA synthase is the enzyme responsible for linking the amino acid to the tRNA • A specific enzyme for each amino acid and not for the tRNA

  35. 2 ‘Adaptors’ Translate Genetic Code to Protein  2 1

  36. Ribosomes • Complex machinery that controls protein synthesis • 2 subunits • 1 large – catalyzes the peptide bond formation • 1 small – binds mRNA and tRNA • Contains protein and RNA • rRNA central to the catalytic activity • Folded structure is highly conserved • Protein has less homology and may not be as important

  37. Ribosome Structures • May be free in cytoplasm or attached to the ER • Subunits made in the nucleus in the nucleolus and transported to the cytoplasm

  38. Ribosomal Subunits • 1 large subunit – catalyzes the formation of the peptide bond • 1 small subunit – matches the tRNA to the mRNA • Moves along the mRNA adding amino acids to growing protein chain

  39. Ribosomal Movement E-site • 4 binding sites • mRNA binding site • Peptidyl-tRNA binding site (P-site) • Holds tRNA attached to growing end of the peptide • Aminoacyl-tRNA binding site (A-site) • Holds the incoming AA • Exit site (E-site)

  40. 3 Step Elongation Phase • Elongation is a cycle of events • Step 1 – aminoacyl-tRNA comes into empty A-site next to the occupied P-site; pairs with the codon • Step 2 – C’ end of peptide chain uncouples from tRNA in P-site and links to AA in A-site • Peptidyl transferase responsible for bond formation • Each AA added carries the energy for the addition of the next AA • Step 3 – peptidyl-tRNA moves to the P-site; requires hydrolysis of GTP • tRNA released back to the cytoplasmic pool

  41. Initiation Process • Determines whether mRNA is synthesized and sets the reading frame that is used to make the protein • Initiation process brings the ribosomal subunits together at the site where the peptide should begin • Initiator tRNA brings in Met • Initiator tRNA is different than the tRNA that adds other Met

  42. Ribosomal Assembly Initiation Phase • Initiation factors (IFs) catalyze the steps – not well defined • Step 1 – small ribosomal subunit with the IF finds the start codon –AUG • Moves 5’ to 3’ on mRNA • Initiator tRNA brings in the 1st AA which is always Met and then can bind the mRNA • Step 2 – IF leaves and then large subunit can bind – protein synthesis continues • Met is at the start of every protein until post-translational modification takes place

  43. Eukaryotic vs Procaryotic • Procaryotic • No CAP; have specific ribosome binding site upstream of AUG • Polycistronic – multiple proteins from same mRNA • Eucaryotic • Monocistronic – one polypeptide per mRNA

  44. Protein Release • Protein released when a STOP codon is encountered • UAG, UAA, UGA (must know these sequences!) • Cytoplasmic release factors bind to the stop codon that gets to the A-site; alters the peptidyl transferase and adds H2O instead of an AA • Protein released and the ribosome breaks into the 2 subunits to move on to another mRNA

  45. Polyribosomes • As the ribosome moves down the mRNA, it allows for the addition of another ribosome and the start of another protein • Each mRNA has multiple ribosomes attached, polyribosome or polysome

  46. Regulation of Protein Synthesis • Lifespan of proteins vary, need method to remove old or damaged proteins • Enzymes that degrade proteins are called proteases – process is called proteolysis • In the cytosol there are large complexes of proteolytic enzymes that remove damaged proteins • Ubiquitin, small protein, is added as a tag for disposal of protein

  47. Protein Synthesis • Protein synthesis takes the most energy input of all the biosynthetic pathways • 4 high-energy bonds required for each AA addition • 2 in charging the tRNA (adding AA) • 2 in ribosomal activities (step 1 and step 3 of elongation phase)

  48. Summary

  49. Ribozyme • A RNA molecule can fold due to its single stranded nature and in folding can cause the cleavage of other RNA molecules • A RNA molecule that functions like an enzyme hence ribozyme name

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