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Different types of rna & translation

Different types of RNA , translation, post translational modification

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Different types of rna & translation

  1. 1. TYPES OF RNA & TRANSLATIO N Dr. Ifat Ara Begum Assistant Professor Dept. of Biochemistry Dhaka Medical College, Dhaka
  3. 3. INTRODUCTION TO RNA one of the three major biological macromolecules that are essential for all known forms of life Other two macromolecules are DNA and proteins
  4. 4. CONTD For many years RNA was believed to have only three major roles in the cell: as a DNA photocopy (mRNA) as a coupler between the genetic code and amino acid, the protein building blocks (tRNA) And as a structural component of ribosomes (rRNA)
  5. 5. CONTD In recent years, however, we have begun to realize that the roles adopted by RNA are much broader and much more interesting RNA has role as enzymes (called ribozymes) to speed chemical reactions. In a number of clinically important viruses RNA (rather than DNA) carries the viral genetic information. 
  6. 6. CONTD RNA has important role in regulating cellular processes (from cell division, differentiation and growth to cell aging and death) Defects in certain RNAs or the regulation of RNAs have been implicated in a number of important human diseases, including heart disease, some cancers, stroke and many others.
  9. 9. 1. MESSENGER RNA A large family of RNA molecules that convey genetic information from DNA to the ribosome, where they specify the amino acid sequence of the protein products of gene expression Single stranded RNA Represent 2-5% of cellular RNA Are found in nucleus & cytoplasm Are produced by post transcriptional modification of primary transcript of a gene (hnRNA)
  10. 10. CONTD It contains only the coding information of the coding region of a gene flanked by 5’ UTR & 3’ UTR
  11. 11. CONTD Monocistronic (one mRNA contains one gene) Half life: short (hours to days) Most heterogenous in size (depends on size of gene) Function: Conveys genetic information from DNA to ribosome & then acts as a template to faithfully translate that genetic information for protein synthesis
  12. 12. 2. TRANSFER RNA An adaptor molecule that serves as the physical link between the mRNA and the amino acid sequence of proteins. It does this by carrying an amino acid to ribosome as directed by a codon in a mRNA. As such, tRNAs are a necessary component of translation
  13. 13. CONTD It represents 10-20% of cellular RNA Smallest RNA found in cytoplasm The structure of tRNA can be decomposed into its primary structure, its secondary structure (usually visualized as the cloverleaf structure), and its tertiary structure
  15. 15. CONTD Attaches with specific AA at its 3’ end Carry anticodon at its anticodon loop Anticodon: A unit made up of three nucleotides that correspond to the three bases of the codon on the mRNA to which it specifically attaches
  16. 16. CONTD  D arm is a 4- to 6-bp stem ending in a loop that often contains dihydrouridine  T arm is a 4- to 5- bp stem containing the sequence TΨC where Ψ is pseudouridine, a modified uridine
  17. 17. CONTD Function: tRNA acts as an adaptor molecule to recognize a definite codon on one hand & a specific AA on other hand. Thus it carries AA to ribosome for protein synthesis
  18. 18. 3. RIBOSOMAL RNA The RNA component of the ribosome In other words, it associates with protein to form ribosome (Structure, which is approximately 60% rRNA and 40% protein by weight. They contain two major rRNAs and 50 or more proteins) It is essential for protein synthesis in all living organisms Represent 70-80% of cellular RNA Found in ribosome & nucleolus
  19. 19. CONTD The ribosomal RNAs form two subunits of ribosome, the large subunit (LSU) and small subunit (SSU) mRNA is sandwiched between the small and large subunits The LSU rRNA acts as a ribozyme, catalyzing peptide bond formation
  20. 20. CONTD Depending on their sedimentation velocity coefficient measured in Svedberg (S) unit, there are 4 types of rRNA I. 28S rRNA II.18S rRNA III.5.8S rRNA IV.5S rRNA Function: rRNA constitutes ribosome which acts as platform where mRNA & tRNA interact for protein synthesis
  21. 21. 4. SMALL NUCLEAR RNA Less than 1% of cellular RNA 30 different types of snRNA exist Function:  Facilitates post transcriptional modification of RNA  Helps in gene regulation
  22. 22. Summary about RNA
  23. 23. Point mRNA tRNA rRNA Conten t 2-5% 10-20% 70- 80% Site Nucleus, Cytoplasm Cytoplasm Ribosome, Nucleolus Size Heterogenous Homogenous Heterogen- ous
  24. 24. Poin t mRNA tRNA rRNA Binds with 40S ribosome AA tRNA & mRNA Func ti-on Acts as template for protein synthesis Carries AA to site of protein synthesis Acts as platform for mRNA & tRNA for protein synthesis
  25. 25. RIBOSOME
  26. 26. WHAT IS RIBOSOME A complex molecular machine found within all living cells, that serves as the site of biological protein synthesis (translation)
  27. 27. CONTD Ribosome links amino acids together in the order specified by mRNA molecules It is made from complexes of RNAs and proteins and is therefore called a ribonucleoprotein Have 2 major components: small ribosomal subunit & large ribosomal subunit . Each subunit is composed of one or more rRNA molecules & a variety of proteins.
  28. 28. CONTD  The small ribosomal subunit: It reads the mRNA  The large subunit: It joins amino acids to form a polypeptide chain The ribosomes and associated molecules are also known as the translational apparatus. Both prokaryotic (E. coli)  and eukaryotic  (human) ribosomes can be broken down into these two subunits
  29. 29. Type Size LSU (rRNA) SSU (rRNA) Prokaryoti c 70 S 50S  (5S : 120 nt, 23S  : 2906 nt) 30S (16S : 1542 nt) Eukaryotic 80 S 60S (5S : 121 nt, 5.8S : 156 nt, 28S : 40S  (18S : 1869 nt)
  30. 30. CONTD Please note, S: Svedberg units nt= length in nucleotides of the respective rRNAs
  32. 32. CONTD  Aminoacyl-tRNA : A tRNA bound to an amino acid  Peptidyl-tRNA: A tRNA containing last AA of the growing peptide chain  [The amino (NH2) group of the aminoacyl-tRNA attacks the ester linkage of peptidyl-tRNA to form a new peptide bond. This reaction is catalyzed by peptidyl transferase]
  33. 33. CONTD  E site : The empty tRNA (that previously was holding onto the last amino acid of peptide chain) is moved to the E site (and what used to be the aminoacyl-tRNA is now the peptidyl- tRNA) Remember: A single mRNA can be translated simultaneously by multiple ribosomes.
  34. 34. TRANSLATIO N
  35. 35. DEFINITION The process in which cellular  ribosomes create proteins Or Synthesis of protein according to the base sequence of mRNA Or mRNA directed protein synthesis where genetic message coded by mRNA is translated in to protein structure
  36. 36. REQUIREMENTS mRNA with initiating codon (AUG coding for methionine) & termination codon (any of 3 stop codons) tRNA AA Ribosome Protein factor: IF, EF, TF (termination factor) ATP & GTP : 2 of each needed for each peptide bond synthesis Amino acyl-tRNA synthetase
  37. 37. AMINO ACYL-TRNA SYNTHETASE Enzyme needed for synthesis of amino acyl-tRNA Catalyzes the attachment of AA with tRNA to form amino acyl-tRNA [In amino acyl-tRNA, the AA is called activated AA & the tRNA is called charged tRNA] Highly selective for a specific AA & its tRNA Has proof reading & editing function
  38. 38. 1. INITIATION Dissociation of 80S ribosome in to 40S and 60S subunit PIC (preinitiation complex) formation: Met-tRNA, IF & GTP binds with 40S ribosome to form PIC
  39. 39. CONTD PIC binds with mRNA Searching for initiating codon (AUG): 40S ribosome scans mRNA from 5’ end towards 3’ end to recognize AUG Synthesis of 80S initiation complex: By PIC+ mRNA+ 60S ribosome
  40. 40. CONTD 60S ribosome containing “P” site and “A” site binds with PIC placing initiator aminoacyl tRNA (met- tRNA) in the “P” site i. P site: Is positioned against initiating codon (AUG) which contains met-tRNA now ii. A site: Is positioned against C1 (1st codon/ the codon that is next to AUG) that is still empty now
  41. 41. CONTD Remember, it is the 60S ribosome on which amino acids are assembled to synthesize protein
  42. 42. 2. ELONGATION OF CHAIN It means simply the ribosome travelling down the message (mRNA) reading codons and bringing in the proper aminoacyl tRNA's to translate the message out to protein. The incoming aminoacyl tRNA is brought into the ribosome A site, where it is matched with the codon being presented
  43. 43. CONTD Done by EF via repeated cycles Successful completion of one cycle translates one codon by recruiting the specified AA of that codon in the process of protein synthesis
  44. 44. CONTD Each cycle can be described under 3 headings: A. Codon recognition B. Peptide bond formation C.Translocation
  45. 45. A) CODON RECOGNITION  Attachment of appropriate amino acyl-tRNA with the empty “A” site positioned against first codon (C1)  Lets think, this amino acid is A1
  46. 46. B) PEPTIDE BOND FORMATION  We know, “P” site positioned against initiating codon (AUG) is already attached with met-tRNA  Methionine (Met) leaves tRNA of “P” site & goes to “A” site  It is followed by formation of peptide bond with the appropriate amino acid (A1) of “A” site  The tRNA of “A” site is now known as peptidyl tRNA (met-A1-tRNA)
  47. 47. C) TRANSLOCATION  Removal of tRNA from “P” site (as this tRNA is empty now) to make the “P” site empty  Then peptidyl tRNA (met-A1-tRNA) moves from “A” site to “P” site to make the “A” site empty  Ribosome moves to next codon down towards 3’ end of mRNA, so that, the loaded “P” site & empty “A” site can be positioned against 1st codon (C1) & 2nd codon (C2) respectively
  48. 48. CONTD Now 2nd cycle of chain elongation begins 2nd cycle translates the 2nd codon (C2) by adding the specific amino acid (A2) of that codon in the growing peptide chain The chain elongation cycle repeats again and again till all the sense codons are translated with the synthesis of a long polypeptide chain
  49. 49. 3. TERMINATION Once a stop codon is reached, the “A” site is positioned against it leading to termination of protein synthesis.
  50. 50. CONTD Facilitated by release factor (RF) / termination factor (TF) The polypeptide is released from the tRNA The tRNA is released from the ribosome and The two ribosomal subunits separate from the mRNA
  51. 51. Post translational modification
  52. 52. INTRODUCTION Chemical modification of proteins  after their biosynthesis Objective: To make the protein functionally active Occurs mostly in ER & golgi apparatus
  53. 53. Protein/PP Foldin g Removal of N- terminal methionine Limited proteolysis e.g. Pepsinogen Intein splicing Chemical/ covalent modificatio n
  55. 55. INTEIN SPLICING An intein is a segment of a protein that is able to excise itself and join the remaining portions (the exteins) with a peptide bond in a process termed protein splicing or Inteins are intervening sequences in certain proteins, comparable to introns in mRNAs (protein introns)
  56. 56. CONTD lnteins have to be removed & exteins ligated in the appropriate order for the protein to become active.
  57. 57. COVALENT MODIFICATION Hydroxylation: Lysine and proline of collagen are hydroxylated after synthesis of collagen Gamma carboxylation: Clotting factors Glycosylation: the addition of a glycosyl group to either arginine, asparagine, cys teine, hydroxylysine, serine, thr eonine,  tyrosine, or tryptophan resulting in a glycoprotein. 
  58. 58. CONTD Phosphorylation:  Addition of phosphate group to a protein (esp. on serine, threonine & tyrosine residue)  Also called “phospho regulation”
  59. 59. WoBBlE HYPotHEsis
  60. 60. INTRODUCTION It explains why multiple codons can code for a single amino acid (degeneracy of genetic code) or It is the phenomenon in which a single tRNA can recognize more than one codon Crick postulated the wobble hypothesis
  61. 61. CONTD One tRNA molecule (with one amino acid attached) can recognize and bind to more than one codon, due to the less-precise base pairs that can arise between the 3rd base of the codon and the base at the 1st position on the anticodon.
  62. 62. CONTD i.e. The pairing between codon (mRNA) and anticodon (tRNA) at the 1st two base position always follows the usual base pairing rule but wobbles (means move a bit) occur at 3rd position
  63. 63. CONTD It reduces the number of tRNA required It accounts for the degeneracy of genetic code (the reason why there is 64 codons but only 40-50 tRNAs)