Fundamental_molecular_biology and genetics.pptx

All molecular biology techniques involved in detection and quantification of gene.
Basis of diagonistic methods

1. Yarsin K LWASA MSc. Biochem. (MUK), MBA (UK) Fundamentals of Genetics And Molecular Biology

2. Reference Textbooks Fundamental Molecular Biology Lizabeth A. Allison, 2007 Edn . Molecular Biology of the Cell Bruce Alberts , 2008 Edn . Molecular Diagnostics for the Clinical Laboratorian William Coleman 2010 Edn .

3. Topical outline Fundamental Molecular Biology Historical perspective Definition and Scope of Molecular Biology Central Dogma of Biology; from structure and functional significance of essential molecules to description of processes therein Signal Transduction Genes, Genomes, Introduction to Bioinformatics Bacterial Genetics Molecular Diagnostics; Definition and Scope Basic techniques in Molecular Biology Molecular Diagnostics Practicum

4. 1. Fundamental Molecular Biology I -Biological molecules -Nucleic acids structure & function -Amino acids & Proteins

5. Objectives To describe the vital Molecules that constitute cells and cellular processes Re-cap: Eukaryotic and Prokaryotic cell structure What defines all life forms? To introduce, define & outline the scope of “Molecular Biology” To describe the “ Central Dogma of Biology ” and the cellular processes therein

6. Fundamental Molecular Biology Outline: Origin of Molecular Biology The Biological Molecules (Biomolecules) Definition and Scope of ‘Molecular Biology’ From Gene to Protein: The Central Dogma of Molecular Biology Nucleic Acids: structure and functional significance DNA Replication, Mutation and repair Transcription Translation The Genetic code, Amino acids, Proteins Genes, Genomes, Introduction to Bioinformatics

7. Origin of Molecular Biology Mendel (1860): Heredity is the transmission of characteristics (gene products) from parent to offspring by means of genes (characters)

8. Origin of Molecular Biology: Classical Genetics Mendel’s experiments Mendel’s laws: Genes (characters) segregate 2. Genes (characters) recombine independently ( Independent assortment ) Mendel (1860): Heredity is the transmission of characteristics (gene products) from parent to offspring by means of genes (characters)

9. Origin of Molecular Biology: Biochemistry Enzymes and catalysis

10. 1928 – 1944: insight into the nature of hereditary material; transforming factor is DNA Griffith, 1928: Genes are DNA Transformation: uptake of naked/free DNA by bacteria First gene transfer mechanism to be discovered Encapsulated Streptococcus pneumoniae : Smooth ( S ), virulent, kills mouse Non-encapsulated: rough ( R ), harmless to mouse Mouse died when injected with mixture of heat-killed S + and live R , but not alone Live S with a capsule was isolated from dead mice A factor from dead S converted live R to S Avery et al, 1944: the transforming factor is DNA! Hence Genes (characters) are DNA!

11. Avery and Griffith: Genes are DNA

12. 1953: the double helix & birth of Molecular Biology

13. Molecular Biology birthed; but what is the pathway of info from DNA to characteristics (phenotype) -Proteins The central dogma of molecular biology : DNA (genes or characters) to mRNA to Proteins (phenotype, characteristics)

14. The Biological Molecules

15. Science > Biology > Medicine Science: The study of “Living” and “Nonliving” things Biology: The study of “Living things” (plants, animals, microorganisms) Life sciences: Biology, Microbiology, Medicine, Immunology, Biochemistry, Cell biology, etc. Medicine is an applied science within biology Other sciences: Chemistry, Physics, Mathematics; (Nonliving things)

16. Biology & the domains of Life The tree of life: what unites all living things?

17. The tree of life based on whole genome data What is common among living things?

18. All Living things have common characteristics What are living things? Anything exhibiting all these 7 characteristics; Reproduction Growth Movement Response to stimuli Excretion Cellular structure Use of energy NB: the characteristics (phenotypes) are encoded by genes (characters)

19. Biological molecules (Biomolecules) Enable life in living things, i.e. life (on earth) depends on these molecules; Nucleic acids (DNA & RNA) Proteins Carbohydrates: energy Lipids/Fatty acids: energy, hormones etc. Water and trace elements: essential

20. Then, what is Molecular Biology? The study Biological Molecules? All life depends on Biological Molecules: Nucleic acids (DNA & RNA) Proteins Carbohydrates: energy Lipids/Fatty acids: energy, hormones etc. Water and trace elements: essential

21. Molecular Biology: definition, scope While life depends on Biological Molecules; Nucleic acids (DNA & RNA) Proteins CHOs: energy Lipids/Fatty acids: energy, hormones etc. Water and trace elements: essential Biological information (i.e. from which characteristics are encoded) is stored and preserved as DNA , and is transmitted into proteins via RNA; hence, Molecular Biology only deals with nucleic acids, proteins and the cellular processes underlying them Molecular Biology: a science that studies how biological information is stored , transmitted and preserved in living things (cells)

22. The Central Dogma of Biology Universal in eukaryotes & prokaryotes Violated by retroviruses (HIV) and other RNA viruses -The flow of biological information from genes (DNA) to proteins via mRNA: DNA → RNA → protein (function/phenotype/characteristic) -DNA makes RNA makes Protein (Wikipedia) Structure Structure Structure Molecular Biology

23. Central Dogma in every aspect of life 23 Replication Function/Phenotype

24. The Central Dogma of Biology Watch!

25. The central dogma of biology

26. The Central Dogma of Biology To appreciate Molecular Biology & related disciplines (e.g. Molecular Diagnostics), you must fully understand the dogma of biology. Focus on; Nucleic acids structure Processes in the dogma: DNA replication, Transcription, Translation. Relate (1) and (2) to Cell structure, eukaryotic and prokaryotic Structure Structure Structure Molecular Biology

27. Re-cup: ultra-cell structure, Prokaryotes vs. eukaryotes 27 Eukaryotes in addition have a cytoskeleton

28. Amazing interconnectedness of eukaryotic membranous structures 28

29. The rough endoplasmic reticulum has ribosomes on its surface and it transports proteins made by the ribosome through the cisternae. Rough endoplasmic reticulum Copyright 1998 Terry Brown. All rights reserved. 29

30. The Central Dogma of Biology To appreciate Molecular Biology & related disciplines (e.g. Molecular Diagnostics), you must fully understand the dogma of biology. Focus on; Nucleic acids structure Processes in the dogma: DNA replication, Transcription, Translation. Relate (1) and (2) to Cell structure, eukaryotic and prokaryotic Structure Structure Structure Molecular Biology

31. Structure of Nucleic Acids, and Functional Significance

32. Nucleic Acids Nucleic acids DNA and RNA Polymers of Nucleotides NB: other biomolecules that are polymers of simple units; Proteins, CHOs, Lipids

33. Nucleic acids structure Primary structure –the components of Nucleic acids The 5-Carbon sugars Nitrogenous bases The Phosphate functional group Nucleotides and Nucleosides The significance of 5’ and 3’ Nomenclature of Nucleotides Secondary structure: the double helix Tertiary structure: Supercoiling

34. Primary structure The 5-Carbon sugars Nitrogenous Bases The Phosphate Functional group Nucleotides and Nucleosides

35. 5-C Sugars in Nucleic Acids The 5-C atoms numbered 1’ to 5’ ; primes are used to distinguish with numbering in nitrogenous bases Both sugars have an O2; 5’-C is outside the ring Sugars differ in Presence or Absence of an O2 in 2’-C

36. The Nitrogenous bases Bases: N2-containing molecules having the chemical properties of a base (a substance that accepts an H+ ion or proton in solution)

37. Before the double helix, Erwin Chargaff; [A] = [T]; [G] = [C] [A] + [G] = [T] + [C] i.e. [Purines] = [ Pyrimidines ] % G + C (base composition of DNA) differs among species but constant in all cells of an organism within a species G + C content varies from 22% - 73%

38. The Phosphate functional group It gives DNA and RNA the property of an acid (a substance that releases a H+ in solution), hence the name nucleic acids

39. Nucleotides and Nucleosides Nucleotides are the monomeric constituents of DNA and RNA. They are composed of; 5-C Sugar Nitrogenous base Phosphate group Nucleosides are composed of; A sugar Nitrogenous base

40. Nucleosides vs. Nucleotides

41. Nucleotides, cont’d Nucleotides are joined (polymerized) by condensation reactions to form chains of DNA and RNA The –OH on the 3’-C of a sugar of one nucleotide forms an ester bond to the phosphate at 5’-C of another nucleotide; The 5’ – 3’ Phosphodiester bond

42. The Phosphodiester bond

43. Nomenclature of Nucleotides -The triphosphate form is the precursor building block for DNA and RNA chain -Knowing complex names of nucleotides is important for understanding scientific literature and for ordering the correct compound when planning an experiment

44. Significance of 5’ and 3’ The two ends of DNA/RNA are designated by symbols 5’ and 3’ 5’ refers to C in the sugar to which a phosphate (PO4) functional group is attached 3’ refers to C in the sugar to which a –OH functional group is attached Thus, symmetry of the end of a DNA strand implies that each strand has a polarity determined by which end bears the 5’-PO4 and which end bears the 3’-OH group

45. Significance of 5’ and 3’ 5’ – 3’ directionality of nucleic acids is an extremely important property. Understanding this polarity is critical for understanding; DNA replication Transcription Reading DNA/RNA sequences Carrying out an experiment in a lab Genomics / Bioinformatics By convention DNA sequences are written with the 5’-end to the left and 3’-end to the right; 5’-TGGCCCGGGTCGACGGTGACACCGTGTTC-3’

46. Secondary structure of DNA: the Double Helix DNA is a double helix; an icon for modern biology Characterized with H-bonding between bases of two strands H-bonding is also referred to as “ Watson-Crick ” or “ Complementary base pairing ” A with T, two H-bonds; G with C, three H-bonds Watson-Crick base pairing explains Chargaff’s rules 1’-C atoms in the two strands are exactly the same distance apart (1.08 nm)

47. Watson-Crick base-pairing in DNA

48. Structure of Watson-Crick double helix Alternating deoxyribose sugars and PO4 groups form the backbone of DNA Bases are attached to sugars, located between the backbones of the DNA strands, lying perpendicular to the long axis of the strands As backbones of the two strands wind around each other, they form a double helix Polarity in each strand is 5’ to 3’ of the double helix; one end has a 5’-PO4, the other end 3’-OH H-bonding occurs only if polarity of the two strands runs in opposite direction; hence the two strands of the double helix are antiparallel Double helix has major and minor grooves

49. The double helix, cont’d

50. http://www.bbc.com/news/science-environment-29760212

51. Significance of complementary bp Ensures storage, preservation and transmission of genetic information ; one strand is coding (sense, non-template strand) , the other is noncoding (antisense, template strand) Ensures fidelity during DNA replication; each of the two daughter DNA molecules has one old strand derived from the parent and one newly made strand; complementary bp results in the two daughter DNA molecules being identical The arrangement of bases determine: Functionality; i.e., genes vs. non-coding DNA Organism or species identity; Evolution Each strand is the predictable counterpart of the other Once you know the sequence of one strand, you can easily figure out the sequence of the other strand; Molecular Biology is the easiest discipline?

52. DNA can undergo reversible strand separation Reversible strand separation allows DNA replication, Transcription, Translation with high fidelity Same features make it possible to manipulate DNA in vitro Unwinding and separation of DNA strands is referred to as “Denaturation” The temperature at which half the bases in a DNA sample have denatured is called Melting Temperature (Tm)

53. Denaturing, Renaturing , Hybridization of DNA

54. DNA tertiary structure: Supercoiling DNA supercoiling is a twisted 3-D structure which is more favorable energetically Supercoiled DNA leads to localized denaturation in which the complementary strands come apart in a short reaction This is important for cellular processes like replication and transcription DNA topoisomerases relax supercoiled DNA

55. Topoisomerases relax supercoiled DNA

56. Drugs targeting DNA topoisomerases Some anti-cancer drugs inhibit topoisomerases Antibacterial agents: DNA gyrase inhibitors Ciprofloxacin and derivatives; Novobiocin , etc.

57. The versatility of RNA

58. RNA: types & structure Polymer of nucleotides What is the difference? Five RNA types: rRNA mRNA tRNA snRNA snoRNA

59. Components of RNA

60. The versatility of RNA RNA folds into unique 3-D structures which act similarly to globular proteins; “ tRNA looks like natures attempt to make RNA do the job of a protein” -Francis Crick Generally the pathway of gene expression from DNA to functional product via an RNA intermediate overemphasizes proteins as the ultimate goal; However, RNAs are involved in a variety of cellular processes along the pathway of gene expression including; DNA replication; RNA processing; mRNA turnover; protein synthesis; protein targeting RNA catalyzes chemical reactions in living cells (ribozymes)

61. Relationship between the 5 RNA types during gene expression

62. tRNA Adaptor like molecule that decodes mRNA codons into amino acids Brings amino acid corresponding to the appropriate mRNA codon Each amino acid has unique tRNA T-loop D-loop Anticodon-loop

63. tRNA structure The three tRNA loops form the cloverleaf secondary structure; each loop has a specific function The T-loop ; involved in recognition by the ribosomes The D-loop ; associated with recognition by the aminoacyl tRNA synthatase The Anticodon loop ; base pairs with the codon in mRNA Every tRNA has the sequence ACC on the 3’-End to which the amino acid is attached The anticodon loop in all tRNAs is bounded by U on the 5’-side and a modified purine on the 3’-side

64. tRNA : secondary and tertiary structures

65. rRNA Type Size Large subunit Small subunit Prokaryotic 70S 50S   ( 5S rRNA ,  23S rRNA ) 30S   ( 16S rRNA ) Eukaryotic 80S 60S   ( 5S rRNA ,  5.8S rRNA ,  28S rRNA ) 40S ( 18S rRNA ) Component of the ribosome, the site of protein synthesis in all living cells Provides a mechanism for decoding mRNA into amino acids and interacts with  tRNAs  during translation by providing  peptidyltransferase  activity RNAs are in parenthesis -Sites of attack for several antimicrobials, especially TB drugs -16S RNA used to classify microorganisms; the Microbiome / Metagenomics

66. Ribonucleoproteins (RNPs) Most RNAs are associated with proteins as RNA-protein complexes called ribonucleotpoteins One important RNP is the ribosome RNA-based catalytic reactions occur in conjunction with proteins Catalytic RNAs are called ribozymes ; they catalyze a number of reactions in cells ranging from cleavage of phosphodiester bonds to peptide formation

67. RNPs are involved in a wide range of cellular processes

68. Types of naturally occurring ribozymes

69. mRNA Information in DNA is copied ( transcribed ) into mRNA, which goes to ribosome for translation mRNA is; -unstable in cells ( why? ) -<5% total RNA -Methylated heads (CH3 cup) - Polyadenylated tails: utilized in biotech -Represents expressed genes in cells Template DNA strand; Antisense, Non-coding strand Reverse Primer Sense; Non-template DNA strand; Coding strand Forward Primer

70. 2. Fundamental Molecular Biology II The processes governing flow of genetic info -DNA replication -Transcription -Translation

71. DNA replication

72. Re-cap; the dogma of biology Each process governing the flow of genetic info is given a specific name; Replication; the process of making an exact copy of DNA from the original DNA Transcription; the process of being copied to generate a ssRNA identical in sequence to one strand in dsDNA The info is rewritten (transcribed), but in basically the same language of nucleotides Translation; the process in which the RNA nucleotide sequence is converted into the amino acid sequence of a protein The term denotes that the info in the language of nucleotides is copied (translated) into another language of amino acids

73. DNA replication The regulation of DNA replication is fundamental to understanding of the continuity of life As cells multiply and give rise to new cells, the genome must be accurately duplicated so that information is passed on to each new generation with minimal error Understanding DNA replication helps to understand molecular tests e.g. PCR, Sequencing

74. DNA polymerases Polymerize nucleotides into growing DNA strands Bacteria have 5 DNA pols, mammals 14 Eukaryotes have 3 DNA pols for chromosomal DNA replication; DNA polymerase α DNA polymerase δ DNA polymerase ε DNA polymerase γ : mtDNA replication The above 4 are called replicative DNA pols ; other types of DNA pols are used in DNA repair processes (e.g. Klenow , etc.; have 3’ – 5’ activity) Chromosomal

75. All DNA pols add nucleotides in the 5’-3’ direction DNA pol catalyzes formation of phosphodiester bond between the 5’C-PO4 of a new dNTP and the 3’C-OH of the last nucleotide in the newly synthesized strand DNA pol cannot initiate DNA synthesis de novo Except for DNA pol α that’s involved in primer synthesis, all DNA pols require a primer DNA pol recognize and bind the free 3’-OH at the end of the primer Once primed, the pol extends pre-existing chains rapidly

76. DNA polymerase works in 5’ – 3’ direction

77. DNA polymerase works in 5’ – 3’ direction

78. 78

79. Semi-conservative nature of DNA replication: gives two daughter duplex DNAs, each with one original strand and one new strand

80. Transcription

81. Transcription The copying of the sequence of the template strand of the gene into a complementary RNA transcript (mRNA) The beginning of gene expression Transcription and translation are coupled in bacteria In eukaryotes, mRNA and protein synthesis are separated between two cellular compartments

82. Transcription and translation coupled in prokaryotes

83. Transcription and translation uncoupled in eukaryotes Regulation at each level

84. Sense and Antisense strands A DNA sequence is read in triplets using the antisense ( non-coding ) strand; Called the template strand , directs the synthesis of RNA via complementary base-pairing The other non-template strand is the sense ( coding ) strand; Bears the same sequence as the mRNA (except for possessing U instead of T)

85. Sense and antisense strands

86. Promoters: in bacteria they have two distinct “Consensus” Sequences A promoters is a sequence to which RNA polymerase binds to begin transcription

87. Structure of bacterial RNA pol Comprised of; Core Enzyme , and A transcription factor called the Sigma factor, δ Together, the core enzyme and δ form a functional enzyme complex called the “ Holoenzyme ”

88. The core enzyme, cont’d The core enzyme catalyzes polymerization It is conserved from bacteria to humans Has high affinity for most DNA; in absence of δ it initiates synthesis anywhere on a DNA template in vitro The δ is responsible for decreasing the non-specific binding affinity of RNA pol

89. The sigma factor ( δ ) Recognizes the promoter The -35 and -10 sequences are required for recognition -10 region is responsible for initial melting of the template strand There several δ per species

90. E. coli sigma factors

91. Types of DNA-dependent RNA polymerases RNA polymerases catalyze RNA synthesis using DNA as a Template; they initiate RNA synthesis de novo (in contrast to DNA pol) Sites for antibiotic attack (RIF, etc.)

92. The mechanism of transcription Occurs in three stages Initiation Elongation Termination

93. Initiation Further divided into three stages; Formation of a closed promoter complex The DNA remains DS and the complex is reversible Formation of an open promoter complex Melting occurs to expose the template strand AT rich -10 region and negative supercoiling helps Irreversible; polymerization is initiated Promoter clearance There is a staged disruption of δ -core enzyme interaction

94. Elongation Transcription bubble is formed as RNA pol winds and unwinds strands The catalytic site of the RNA pol has both; A Substrate Binding Sub-site at which the incoming NTP is bound to the pol and to the complementary nt residue of the template And a Product Binding Sub-site , at which the 3’-terminus of the growing RNA chain is positioned NTP and a phosphodiester bond forms with the 3’-OH of the last nt in the RNA chain Transcription also proceeds in 5’ – 3’

95. Transcription elongation

96. Termination The RNA pol moves down the DNA until a stop signal or terminator sequence is reached Two types of terminators; Rho dependent Rho independent All recognize inverted repeats that form stem-loop structures

97. Gene expression regulation Except the house keeping genes, not all genes are expressed all at once Certain gene products (proteins) regulate / control expression of the genes The “Operon” model by Jacob & Monod led to the discovery of mRNA Read the Lac, Try and Arabinose operons Signal transduction: regulates gene expression mostly in eukaryotes Quorum Sensing / Cell-Cell Signalling : regulates gene expression in bacteria and some unicellular protozoa (African Trypanosomes)

98. Translation

99. Translation The process in which the RNA nucleotide sequence is converted into an amino acid sequence of a protein The term denotes that the info in the language of nucleotides is copied (translated) into another language of amino acids Translation occurs at the ribosomes, and understanding their structures is key

100. Two functions of ribosomes Decoding the genetic code in the mRNA Catalyzing the formation of the peptide bonds between amino acids resulting in a polypeptide chain In essence, a ribosome is an enzyme, A Polypeptide Polymerase

101. Structural significance of ribosomes Ribosomes have two subunits, large and small Prokaryotic: 70S , comprising of 50S & 30S Eukaryotic: 80S , comprising of 60S & 40S The Peptidyl Transferase center and the catalytic site are in the large s/unit (60S / 50S) The small subunit serves as the assembly guide for factors needed in protein synthesis; decoding the mRNA also occurs on the small subunit (40S / 30S)

102. Recap; Components of Ribosomes

103. Ribosome binding sites Ribosomes have 3 tRNA -binding sites The Acceptor site (A) The Peptidyl site (P) The Exit site (E) Each of the above is occupied in succession by a particular tRNA during protein synthesis cycle

104. A-site: occupied by aminoacyl- tRNA , i.e. the charged tRNA P-site: occupied by peptidyl- tRNA , i.e. the tRNA carrying the growing peptide chain. The P-site is also referred to as the Puromycin sensitive site. Puromycin is an antibiotic which shows similarities with a part of aminoacyl- tRNA . E-site: the ribosomal site harboring decylated tRNA on transit out from the ribosome.

105. Ribosome binding sites P site A site E site 40S/30S (small subunit) 60S/50S (large subunit) Anticodon

106. tRNAs bridge the large and small s/units; With the anticodon arm of tRNA pointing towards the small s/unit for decoding And the acceptor arm of tRNA pointing into the large s/unit for peptidyl transferase Ribosomes function in the cytoplasm, but their assembly occurs in the nucleolus

107. The nucleolus and ribosome biogenesis The nucleolus is the site of rRNA synthesis and ribosome assembly Except for 5S rRNA , rRNA genes are transcribed by RNA pol I into one long precursor rRNA 5S rRNA is transcribed from a separate gene by RNA pol II Eukaryotic large and small ribosomal s/units are assembled in the nucleolus before export to the cytoplasm

108. Aminoacyl-tRNA synthetases The fidelity of translation is dependent on the accuracy of two processes; Codon-Anticodon recognition Aminoacyl-tRNA synthesis Aminoacyl-tRNAs are synthesized by the 3’-Esterification of tRNAs with the appropriate amino acid; Aminoacyl-tRNA synthetases catalyze the reactions The uncharged tRNA is aminoacylated to generate a charged tRNA , which then interacts with the elongation factor ( eEF )

109. Aminoacyl-tRNA charging Aminoacyl- tRNA synthetases attach amino acids to tRNAs in two enzymatic steps; Amino acid reacts with ATP to become adenylated (addition of AMP). Amino acid is attached by a high energy ester bond between the carbonyl group of the amino acid and the phosphoryl group of AMP AMP is released and the amino acid is transferred to the 3’-end of tRNA to form charged tRNA Specific aminoacyl-tRNA synthetases are denoted by their 3 letter amino acid designation Met RS ; Methyl- tRNA synthetase ( Enzyme ) tRNA met ; Uncharged tRNA specific for Methionine Met - tRNA ; tRNA aminoacylated with Methionine ( Charged tRNA ) Each of the 20 amino acids has a specific aminoacyl-tRNA synthetase

110. Activated/charged tRNA Two steps in aminoacyl tRNA charging

111. The mechanism of translation Also occurs in three stages; Initiation Elongation Termination Each step involves multiple factors and energy from GTP hydrolysis

112. Initiation The most complex, most tightly controlled stage The ribosome is assembled at the Initiation Codon in the mRNA with a Methionyl Initiator tRNA bound to its P site Initiation further subdivided into; Formation of Ternary Complex and Loading onto the 40S s/unit Loading the mRNA on the 40S s/unit Scanning the start codon recognition Joining of the 40S and 60S s/units to form the functional 80S s/unit

113. Ternary complex formation and loading onto the 40S s/unit Assembly of the ternary complex is the 1 st step in the initiation pathway Ternary complex is comprised of eIF2 , GTP , and the amino acid-charged initiator tRNA ( Met- tRNA ) The complex binds to the 40S s/unit to form a 43S complex

114. Loading the mRNA on the 40S s/unit In bacteria (coupled transcription/translation) soon as Shine- Dalgano emerges from the transcriptional apparatus, it is bound by the 30 small s/unit Eukaryotic mRNA to be translated is fully processed; Spliced , 5’-capped , poly-A tailed The 5’-end of mRNA is identified by the m 7 GpppN cap IFs associate with 5’-cap, unwind 2 o and 3 o structures and remove RNA binding proteins Other IFs associate with poly A binding protein bound to the 3’-poly A tail Poly A tail recruits the 43S complex to the mRNA and when bound with PABP, signals translation Together, the 5’ and 3’ IF complexes work to load the mRNA onto the 43S complex

115. Scanning and AUG recognition Once the mRNA is loaded, the 43S complex scans along the message from 5’ to 3’ looking for AUG start codon Once the 43S complex encounters an AUG codon (usually 1 st AUG) embedded in the kozak consensus sequence; a stable 48S complex is formed Upon arrival at AUG, codon-anticodon interaction occurs by complementary bp with an antiparallel orientation between tRNA and mRNA

116. Joining of the 40S and 60S s/units Initiation involves two GTP hydrolysis events; One catalyzed by eIF2 upon AUG recognition Another at the end of the pathway, after 80S complex formation

117. Translation initiation

118. Elongation Similar in prokaryotes and eukaryotes Occurs rapidly; aminoacyl tRNA enter the A site where decoding occurs If they are the correct ( Cognate ) tRNAs , the ribosome catalyzes formation of a peptide bond between the incoming aa and the growing polypeptide chain After the tRNAs and the mRNAs are translocated such that the next codon is moved to the A site, the process is repeated

119. Elongation, details Peptide chain elongation begins with a peptidyl-tRNA in P site next to a vacant A site An aminoacyl-tRNA is carried to the A site as part of a ternary complex with GTP and eEF1A Cognate (correct) codon-anticodon bp causes 3 bases in the 18S rRNA to swing out and interact with the resulting mRNA- tRNA duplex This activates the GTPase activity of eEF1A eEF1A GDP releases the aminoacyl-tRNA into the A site in a form that can continue with peptide bond formation

120. Peptide bond formation and translocation The ribosomal peptidyl transferase center catalyzes formation of a peptide bond between the incoming amino acid and the peptidyl-tRNA The resulting deacylated tRNA is moved into the E site of the large s/unit, the peptidyl-tRNA is moved into the P site, and the mRNA moves by 3 nt to place the next codon of mRNA into the A site Translocation is mediated by eEF2 and requires GTP

121. The 23S and 28S rRNAs are catalytic Peptide bond formation is catalyzed by 23S / 28S rRNAs , the ribozymes Discovered in 2000, proving that ribosomal RNA is catalytic Prior to the year 2000, only proteins were thought to be the enzymes (biological catalysts)!

122. Termination The ribosomal peptidyl transferase center is responsible for two fundamental reactions; Peptide bond formation, and Nascent peptide release during elongation and termination phases of protein synthesis Translation termination occurs in response to presence of a stop codon ( UAG, UAA, UGA ) at the A site The end result is the release of the completed polypeptide following hydrolysis of the ester bond linking the polypeptide to the P site tRNA Termination requires two release factors; Class I RFs decode stop codons Class II RFs are GTPase

123. The Genetic code, Amino acids and Proteins

124. The Genetic Code Provides fundamental rules for decoding genetic info into polypeptides by way of translation 64 three letter codes ( codons ) Codon: 3-base sequence in mRNA which specifies a single aa to be added into a polypeptide chain or causes termination of translation Anticodon: 3-base sequence in tRNA that bp with a specific codon in mRNA 61 codons are recognized by the 20/2 specific tRNAs for incorporation of 20/2 amino acids Start codons: AUG in genes of most species GUG and UUG also used in 15% bacterial genes Stop codons: UAA , UAG , UGA Also code for Selenocysteine and Pyrrolysine in archae

125. Cracking the genetic code

126. The genetic code is degenerate All codons are degenerate except those encoding M and W, i.e., tRNAs specific for a particular aa respond to multiple codons that differ only in the 3 rd letter, hence; “Wobble” hypothesis The pairing between two codons and anticodons at 1 st two codon positions always follows rules of complementary bp , but exceptional “wobbles” (non-Watson-Crick bp ) can occur at the 3 rd position Initially the genetic code was thought to be universal; but in certain organisms and organelles the meaning of some codons has changed; CUG for S in C. albicans , UGA for W in mitochondria, Bacillus subtillis

127. Common alternative meaning of codons

128. Codon bias/usage The frequency with which degenerate codons are used varies significantly across species and between proteins expressed at high or low levels within the same organism E.g. AGG and AGA for R in mammals is rarely used in E. coli ; tRNA Arg that reads infrequently used AGG and AGA codons for R is present at extremely low levels in E. coli – genes wont express! Redesign the gene or host for optimal expression; codon-optimization

129. Proteins and structure Whereas DNA is composed of only 4 nt , proteins contain 20 common aa , at times 22 Some proteins contain an abundance of one amino acid, while others lack one or two types of amino acid Selenocysteine (from which Selenium is obtained) and Pyrrolysine are the 21 st and 22 nd amino acids, respectively, found in Archaea

130. Amino acids Each amino acid has; H NH 3 + COO-, Side chain (R) All attached to one α -C atom The difference between any two aa is their R groups; Each R group has distinct properties such as charge, hydrophobicity, polarity It’s the arrangement of aa, with their distinct R groups, that give each protein its xtc structure and function α -C atom

131. The 20 genetically encoded amino acids

132. The 22 genetically encoded amino acids and their properties

133. Primary structure of proteins Amino acids joined together by peptide bonds The NH3+ of one molecule reacts with the COO- of the other to form a peptide bond Peptide: short sequence of amino acids Polypeptide: longer chains of aa of known sequence and length When joined in series of peptide bonds, aa are called residues to distinguish btn the free form and the form found in proteins Polypeptide chain has polarity and by convention is depicted with the free NH3+ at its left (i.e. N-terminus ) and the free COOH at the right ( C-terminus ) As in DNA and RNA sequences, aa are read from left to right The individual aa have 3 letter and single letter abbreviations

134. Peptide bond formation

135. Secondary structure Interactions of aa with their neighbors gives a protein its 2 o structure The interactions are stabilized by H-bonds , disulfide bridges , hydrophobic contacts , van der Waals interactions , H-bonds btn non-backbone groups and electrostatic interactions 3 basic elements of protein 2 o structure: α -helix , β -pleated sheet , and the unstructured turns that connect the elements

136. α -Helix α -helices are stabilized by H-bonding among near neighbor aa with each residue being H-bonded to two other residues Approx. 30% of residues in globular proteins are found in α –helices Most aa contribute to α –helical structure except P

137. β -pleated sheet Involves extended aa chains in a protein that interact by H-bonding The chains are packed side by side to create a pleated, accordion-like appearance Two segments of a polypeptide chain (or two individual polypeptide chains) can form two different types of β –structures

138. Turns Connect the α –helices and the β –pleated sheet elements in a protein

139. Tertiary structure The folded 3-D shape of a polypeptide is its tertiary structure; the functional state of most proteins Tertiary structures are stabilized by covalent and non-covalent bonds but mostly the latter Covalent bonds within and between polypeptides are disulfide (S-S) bonds or bridges btn Cysteines

140. Protein structures

141. The three main categories of 3 o structure are Globular, Fibrous and Membrane proteins Globular proteins are the majority Oval in shape e.g. lysozyme Fibrous proteins are filamentous or rod-like Are the structural components of skin, tendons, ligaments, teeth, bones. e.g. α –keratins in hooves, nails, hair; and actin in the cytoskeleton Membrane proteins are TM helical proteins traversing the cell membranes. Mostly 7 pass TMD and have predominant hydrophobic residues e.g. G-protein receptors

142. Examples of Globular, Fibrous, and Membrane proteins

143. Quaternary structure A functional protein can be composed of one or more polypeptides, forming a Quaternary structure The term s/unit is used to refer to individual polypeptide chains Quaternary structures can be based on protein with identical s/units or non-identical s/units The presence of this higher order structure allows versatility in function i.e. catalytic or binding sites are often formed at interface btn s/units E.g. Hb : a tetramer of two different s/units that join to form a binding site for a heme group; two alpha and two beta s/units i.e. α 2 β 2 Antibodies contain two heavy and two light chains with the antigen binding site formed by the interaction of the two chains