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Topics: 3.5, 7.3 & 7.4

Topics: 3.5, 7.3 & 7.4. PROTEIN SYNTHESIS Cells require proteins to carry out activities Enzymes, tranport oxygen, carbon dioxide Cells must produce these proteins when required  PROTEIN SYNTHESIS. Vernon Ingram 3.5.5 One Gene - One Polypeptide Hypothesis.

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Topics: 3.5, 7.3 & 7.4

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  1. Topics: 3.5, 7.3 & 7.4 PROTEIN SYNTHESIS Cells require proteins to carry out activities Enzymes, tranport oxygen, carbon dioxide Cells must produce these proteins when required  PROTEIN SYNTHESIS

  2. Vernon Ingram 3.5.5 One Gene - One Polypeptide Hypothesis

  3. Genes not only code for enzymes, but code for proteins, such as hemoglobin, that are made up of more than one polypeptide. • Significance of Ingram's findings: he linked a human abnormality (sickle cell disease) to a single alteration in the amino acid sequence of the hemoglobin protein.

  4. 3.5: Protein Synthesis Overview

  5. The Central Dogma • ·The central dogma of molecular biology deals with the detailed residue-by-residue transfer of sequential information. • ·It states that information cannot be transferred back from protein to either protein or nucleic acid. DNA  RNA  protein • DNA cannot leave the nucleus (keeps it from being damaged)

  6. Protein Synthesis The majority of genes are expressed as the proteins they encode. The process occurs in 3 steps: Transcription (DNA---> RNA) Translation (RNA ---> protein) Termination

  7. Transcription in Eukaryotesie: Animal cells

  8. Protein Synthesis in Prokaryotesie: single celled Bacteria

  9. Where does all this Happen?

  10. Overview of Protein Synthesis

  11. Protein Synthesis • Nucleic Acids (DNA & RNA) carry the hereditary information. • This information is contained in codons. • What are Codons? A codon is a set of three base pairs in mRNA (A, T, C, G) that directs or codes for amino acids. (ex. GCU = Alanine)

  12. 3.5.3 THE GENETIC CODE • There are 20 amino acids, but only four different nitrogenous bases. To code for all amino acids, a sequence of three nucleotides must be used for each amino acid. • Each triplet is called a codon • Because there are 64 different arrangements of the four possible bases in groups of codons, and there are only 20 amino acids, some amino acids have several different codes, resulting in some redundancy in the genetic code. •   One codon serves as the start codon and others serve as the stop codons.

  13. Stage 1: TRANSCRIPTIONDNA codes for RNA copy! • DNA is used to produce: • mRNA (messenger RNA) • tRNA (transfer RNA) • rRNA (ribosomal RNA)

  14. RNA • Contains instructions for making proteins. • Made up of four nucleotides acting as counterparts to the DNA. • Consists of a single long chain of nucleotides. • Made up of Adenine, Uracil, Cytosine and Guanine.

  15. 3.5.1 Comparing RNA and DNA

  16. 3.5.2 Phase 1: Initiation • RNA polymerase binds to a region of the DNA (upstream) adjacent to the start of a gene known as the promoter region (recognition site). • Promoter region consists of adenine and thymine bases – TATA Box. • DNA is unwound separating the template strand and the coding strand.

  17. TATA Box

  18. 7.3.1 Phase 2: Elongation • Using the template strand, RNA polymerase builds the complementary mRNA molecule in the 5’ – 3’ direction. • Each nucleotide is added by removing 2 phosphate groups from the nucleoside triphosphate and forming a phosphodiester linkage. (similar to DNA replication) • The coding strand is not used for this process and is identical to the mRNA except it contains uracil.

  19. 7.3.2: Sense and anti-sense • The sense strand has the same base sequence as the transcribed mRNA except that T is replaced by U • The anti-sense strand acts as the template for the transcription of mRNA • The RNA nucleotides are polymerized along the sugar phosphate backbone by RNA polymerase

  20. DNA Strand 1: AGCTATCGAGCAT DNA Strand 2: TCGATAGCTCGTA RNA copy: AGCUAUCGAGCAU • Transcription stops when the termination sequence is reached. • The completed RNA copy is now called messenger RNA or mRNA and carries the coded message to the ribosomes in the cytoplasm.

  21. 7.3.3 Phase 3: Termination • RNA polymerase recognizes the terminator sequence at the end of the gene. • The sequence differs between prokaryotes and eukaryotes. • Transcription ceases, mRNA dissociates from the DNA Template and RNA polymerase is free to transcribe another gene.

  22. Transcription

  23. 7.3.4: RNA Processing • Prior to leaving the nucleus of eukaryotic cells, RNA known as the primary transcript goes through a series of modifications. • Primary RNA must be processed before leaving the nucleus in order to be protected. • This is done through capping and tailing. • Spliceosomes also cut out introns and remaining exons are joined together to form the final mRNA.

  24. Posttranscriptional Modifications 1. Capping • 5’ cap consisting of 7-methyl guanosine units is added. • Protects the mRNA from digestion from nucleases and phophatases. • Involved with the initiation of translation

  25. Capping

  26. 2. Tailing • A poly-A tail is added to the 3’ end. • Consists of approximately 200 - 300 adenine ribonucleotides added by poly-A polymerase. • Protection from degradation, facilitates attachment to ribosomal complex, assists in export of nucleus) .

  27. Tailing

  28. 3. Removal of Non-Coding Regions • Exons – Coding regions are interrupted by… • Introns – Non-coding • Spliceosomes cut out the introns and rejoin the exons.  RNA splicing • small nuclear ribonucleoproteins (SnRNPs) play a key role in RNA splicing

  29. RNA Modification

  30. Why have introns? • ·Intron DNA sequences may control gene activity • ·The splicing process may help regulate the export of mRNA • ·Introns may allow a single genet to direct the synthesis of different proteins (i.e if the same RNA transcript is processed differently)

  31. mRNA Transcript • After the primary transcript has been capped, tailed and spliced, it is now known as mRNA transcript.

  32. 3.5.4/ 7.4.6 Stage 2: Translation: mRNA ---> Polypeptide

  33. mRNA • Processed transcript with 5’cap, poly-A tail and introns (non-coding regions) removed.

  34. 7.4.1tRNA • Each tRNA is specific for amino acids, therefore there are 64 types of tRNA since there are 64 possible codons. • It is proposed that tRNA can recognize more than one codon by unusual pairing. This is known as the wobble hypothesis/degenerate code. • tRNA with attached amino acid = aminoacyl-tRNA (charged)

  35. Specificity • The shape of each tRNA is different • tRNA selects a specific enzyme (aminoacyl-tRNA-synthetase) which adds a specific amino acids to the base sequence at the 3’ end of the tRNA *** ATP required

  36. 7.4.2 Ribosomes ·are made up of two subunits (60% rRNA and 40% protein) that hold the mRNA in place in order for translation to occur. ·Each ribosome has three biding sites: ·P site = holds tRNA carrying growing peptide ·A site = holds tRNA carrying next amino acid ·E site = where tRNA exits from

  37. 7.4.3 Phases of Translation • Initiation • Elongation • Translocation • Termination

  38. 1.Translation = synthesis of a polypeptide, which occurs under the direction of mRNA ·Linear sequence of bases in mRNA is translated into the linear sequence of amino acids ·Translation occurs at protein-synthesizing machinery, which consists of ribosomes, ribosomal RNA (rRNA), and proteins that facilitate the addition of amino acids to form polypeptide

  39. 7.4.4 Phase 1 - Initiation • mRNA is sandwiched between the two ribosome subunits. • Ribosome reads downstream (5’  3’) until it reaches the start codon-AUG • The corresponding tRNA carrying methionine binds to the P (peptide) site.

  40. Phase 2 - Elongation • The next tRNA carrying the required amino acid enters the A site. • The amino acids are joined by peptide bonds. • tRNAs are released through the passive site and are recycled.

  41. 7.4.5 Peptide bonds

  42. Phase 3: Translocation • The mRNA is moved along one codon and thus the ribosome has moved one codon in a 5’-3’ direction. • The first amino acids is released from its tRNA. • The first tRNA is now in the third binding site of the ribosome (E-site). • The second tRNA is now in the middle position (P-site) and the A-site can receive a third tRNA with an anti-codon that fits to the new codon on the mRNA.

  43. Phase 3 - Termination • Ribosome reaches a stop codon (UGA, UAG or UAA) • Release factor proteins release the polypeptide chain. • Ribosome spits into its subunits. • Ribosome and mRNA are recycled. • Polypeptide undergoes modification. • http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter15/animations.html#

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