Lecture 1.pptx
- 1. BME 403: Molecular Biology for Engineers The Central Dogma, DNA, RNA Lecture 1 Akid Ornob
- 2. Objective - The Central Dogma
- 3. Cells – Basic unit of life • Cells – basic unit of a living organism • It contains hereditary information which it passes on to the next generation • Cells gather raw materials from its environment • And takes decisions through a complex networks of chemical reactions known as pathways • > 10^13 cells in an average human
- 4. Cells Prokaryotic cell Eukaryotic cell 1. Eukaryotic cells have nucleus that encapsulates their DNA 2. Prokaryotic cell do not have nucleus 3. Eukaryotic cells contain membrane-bound organelles
- 5. Nucleus and DNA packaging Chromatin carries DNA and histone protein
- 6. 6 Adenosine Mono Phosphate - AMP -H in DNA base sugar phosphate AMP is a nucleotide building block (dAMP for DNA/RNA) The structure of nucleic acids (DNA/RNA)
- 7. 7 Uracil in RNA Bases for DNA are shown above. RNA has uracil instead of thymine. Nucleotides are - for RNA or DNA Adenosine Guanosine Uridine Cytidine Deoxyadenosine Deoxyguanosine Deoxythymidine Deoxycytidine The chemical nature of polynucleotide
- 8. Nucleosides Carbon numbering of sugars in nucleosides is important
- 9. • Ordinary numbers are used in bases • Primed numbers are used sugars • Base is linked to 1’ position • 2’ position is deoxy in deoxynucleosides • Sugars are linked together in DNA and RNA through their 3’ and 5’ -positions Carbon numbering in nucleosides
- 10. Nucleotides (Sugar + Base + phosphate) • A nucleotide is a nucleoside with a phosphate group attached through a phosphodiester bond
- 12. 12 Watson-Crick basepairing scheme Hoogstein basepair scheme Protonated C + Base-pairing in DNA In double-stranded DNA: A always bonds to T C always bonds to G The bases are held by hydrogen bonding - 2 H bonds between A and T - 3 H bonds between C and G
- 13. DNA as a double helix Exists as a double helix 2 strands of DNA running anti-parallel to each other (one 5’-3’ and the other 3’-5’) The DNA sequence is read from 5’-3’
- 14. DNA replication DNA replication is semi-conservative
- 15. DNA replication – interplay between enzymes DNA replication occurs prior to cell division and is essential transferring the genetic information to the next generation
- 16. Mutation • Accuracy of DNA replication is extraordinary • Several proofreading mechanisms are used • DNA is copied with fewer than one mistake in 109 nucleotides added • Very rarely, the replication skips or adds a few nucleotides, or puts a T instead of C or A instead of G • Any change of this kind is a genetic mistake, call a mutation.
- 17. Effect of Mutation • Mutation cause alteration of gene • May lead to inactivation of a crucial protein • Result in cell death, mutation will be lost • Mutation may be silent • Very rarely, improved gene with novel function • In this case mutation will be carried to next generation • May lead to uncontrolled cell division and the formation of a tumor • Tumor may be malignant and cause cancer
- 18. DNA Repair • The altered portion is recognized and removed by enzymes called DNA repair nucleases
- 19. The chromosome • DNA is packaged into individual chromosomes • Prokaryotes typically have a large single chromosome (e.g. bacteria, archea) • Eukaryotes have species specific number of linear chromosomes (e.g. animals, plants) • A growing field of research in genetics and bioinformatics is epigenetics – interaction of the genetic code with the histone protein and its effects on the phenotype of a cell
- 20. Gene and the genome • Genome – complete set of DNA for a given species • Human genome -> 23 pairs of chromosomes • All cells except for the sex cells and mature red blood cells contain the complete genome • Genes – basic unit of heredity • Contains a sequence of nucleic acid bases that encodes the information required to synthesize a particular protein • Human genome consists of ~25,000 protein coding genes
- 21. Gene density Not all of the DNA in a genome encodes protein Bacteria – 90% coding gene/kb Human – 1.5% coding gene/kb (Total genetic material = 3Gb) • Eukaryotic genes are broken into pieces called exons • Separated by seemingly meaningless piece called introns • Prokaryotic genes are continuous
- 22. Gene Sequence Determines Protein Amino Acid Sequence
- 23. 23 Sugar (ribose) Pucker The critical difference in RNA and DNA double helix 3’ -OH in RNA stabilizes C-3’ endo ribose configuration RNA B-DNA
- 24. 24 Sugar Pucker The sugar ring has different conformations for RNA and DNA Note the locations and distances of phosphate groups in RNA and DNA differ due to change in sugar ring conformation RNA B-DNA B-DNA RNA (A-DNA) Substantial consequences Backbone is more extended In DNA
- 25. 25 Double-stranded DNA structure A. Initially deduced from low resolution fiber diffraction studies (X-ray on fibers) B. High resolution structure from crystals of short DNA oligomers -much more detail -base pair variations, structural variations (B-DNA, A-DNA, Z-DNA)
- 26. 26 Double-strand DNA/RNA Fiber Diffraction Studies: 3 major families B-family A-family Z-family B RNA left handed helix C RNA•DNA D DNA B-DNA: standard form of double helix of DNA A-DNA: induced by binding of eukaryotic transcriptional promoter (TATA binding protein) to DNA Z-DNA: May form in regions of the genome with dinucleotide repeats (CA)n or (CG)n
- 27. 27
- 28. 28 DNA is polymorphic-environmental conditions can alter structure “Phase diagram” relating conformational changes of natural DNAs with different (dG+dC) content with varying relative humidity.
- 29. 29 A-DNA is like double-stranded RNA B-DNA is the predominant form of double-strand DNA
- 30. 30
- 31. 31 The phosphate groups of A-DNA and Z-DNA can be “economically” hydrated by a single bridging water - this is why these DNA forms are favored at low hydration High salt also favors Z-DNA based on electrostatics from phosphate-repulsion (PNAS 1993 90, 5740) Comparison of deoxyribose-3’, 5’-diphosphate fragments in a, A-, b, B- and c, Z-DNA. Vertical lines represent helical axes; phosphorous atoms indicated by shading, phosphate oxygens by numbering. Shortest distances between free phosphate oxygen atoms are give in Å, W represents water molecules that bridge free phosphate oxygens in A- and Z-DNA. Capital letters B in A- and B-DNA and G, C in Z-DNA indicate C(1’) sugar atoms where bases are attached Nature 1986 324, 385- A-DNA B-DNA Z-DNA
- 32. 32 Z-DNA-left handed helix Defined by crystal structure of d(CpGpCpGpCpG) -12 bp/turn -alternating syn anti base configuration -major groove “filled” (only minor groove) Can be formed when (GC)n tracts are present in DNA sequence Methylation of cytosine favors B Z Biological relevance??? Still not clear. May be present in stretches of repeating dinucleotides with alternating purine/pyrimidine
- 33. 33 Z-DNA vs B-DNA CpGpCpGpCpG (left handed) (right handed) Van der Waals side views of Z-DNA and B-DNA. Two views of Z-DNA are shown which are 30o apart in orientation about the helix axis. Zig-Zag backbone Z-DNA B-DNA
- 34. 34 dsDNA reversibly self-assembles at physiological temperature, pressure, pH, and ionic composition: Gds < Gss
- 35. 35 Thermal Denaturation of dsDNA
- 36. DNA vs RNA • Genetic information encoded in a particular gene (strand of DNA) is transferred to RNA via transcription • RNA retains all the information of DNA sequence • However the differences are • RNA contains ribose instead of deoxyribose sugar • The base thymine (T) is replaced by uracil (U) • RNA is usually single stranded and structurally more versatile than DNA
- 37. • Ribonucleic acids play three well-understood roles in living cells: • Messenger RNAs encode the amino acid sequences of all the polypeptides found in the cell • Transfer RNAs match specific amino acids to triplet codons in mRNA during protein synthesis • Ribosomal RNAs are the constituents of ribosomes and catalyzes protein synthesis • Ribonucleic acids play several less-understood functions in eukaryotic cells: • MicroRNA appears to regulate the expression of genes, possibly via binding to specific nucleotide sequences • Ribonucleic acids act as genomic material in viruses Overview of RNA function
- 38. • Ribonucleic acids are synthesized in cells using DNA as a template in transcription • Transcription is tightly regulated in order to control the concentration of each protein • Being mainly single-stranded, many RNA molecules can fold into compact structures with specific functions • Some RNA molecules can act as catalysts (ribozymes), often using metal ions as cofactors • Most eukaryotic ribonucleic acids are processed after synthesis • Elimination of introns; joining of exons • Poly-adenylation of the 3’ end • Capping the 5’ end Overview of RNA metabolism
- 39. Initiation: - RNA polymerase binds to sequence called promoter to begin transcription. DNA double helix unwinds - RNA polymerase covers about a 35 bp-long segment of DNA Elongation: - RNA polymerase unwinds from the promoter region and adds nucleoside triphosphates add to the 3’ end of the growing RNA strand - The growing chain is complementary to the template strand of the DNA Termination: - RNA transcription ends when the RNA Polymerase encounters a terminating signal in the DNA -> formation of an RNA “hairpin” structure which destabilizes the interaction between the Polymerase and the RNA - The transcribed mRNA is released Mechanism of transcription in E.Coli
- 41. Template and Coding strands • DNA Template Strand – serves as template for RNA polymerase • DNA Coding Strand – the non-template strand; has the same sequence as the RNA transcript
- 42. mRNA processing • Introns cleaved • Exons spliced together • Poly-A cap on the 3’ end (stabilizes the mature mRNA structure)
- 43. Translation and Protein Biosynthesis Protein life cycle in eukaryotes
- 44. • There are 20 common, genetically encoded amino acids • A four-letter code in groups of two is insufficient (16) • A four-letter code in groups of three IS sufficient (64) • Living organisms use non-overlapping mRNA code with no punctuation The genetic code for proteins consists of triplets of nucleotides
- 45. Overlapping vs Non-overlapping code
- 46. Reading Frames • Reading frame is set at the initiation of translation process • In almost every case, only one reading frames will produce a functional protein • This reading frame is known as open reading frame
- 47. • The code is written in the 5’ → 3’ direction • Third base is less important in binding to tRNA • First codon establishes the reading frame • If reading frame is thrown off by a base or two, all subsequent codons are out of order • 61/64 codons code for amino acids • Three are termination codons • UAA, UGA, UAG • AUG = initiation codon (as well as Met codon) Features of the genetic code
- 49. Genetic code is degenerate • 4 nucleotides; 43=64 codons possible • 3 sequences for stop codon, 1 for start (also Met) • 61 different codons to specify only 20 amino acids • Only Met and Trp have unique codons • Either each amino acid is specified by >1 tRNA or tRNAs can bind to more than one codon (Both occur)
- 50. Genetic Code • Amino acid sequence of protein is constructed through the translation of information encoded in mRNA • Amino acids are specified by mRNA codons consisting of nucleotide triplets. Translation requires adapter molecules, the tRNAs, that recognize codons and insert amino acids at the correct position in the protein
- 51. Genetic code (cont.) • AUG signals initiation, and UAA, UAG, and UGA signal termination • Genetic code is degenerate—multiple code words for almost every amino acid • Standard genetic code words are universal for almost every species • Third position in each codon is less specific than the first two
- 53. Adapter (tRNA) brings amino acid to mRNA
- 54. • The codon sequence is complementary with the anticodon sequence • The codon in mRNA base pairs with the anticodon in mRNA via hydrogen bonding • The alignment of two RNA segments is antiparallel Molecular Recognition of Codons in mRNA by tRNA
- 55. • ssRNA of 73–93 nucleotides in both bacteria and eukaryotes • Cloverleaf structure in 2-D • “Twisted L” shape in 3-D • Most have G at 5’-end; all have CAA at 3’-end • Amino acid arm • Has amino acid esterified via carboxyl group to the 2’-OH or 3’-OH of the A of the terminal CAA codon Structure and characteristic features of tRNA
- 56. • Anticodon arm Have modified bases Methylated bases, etc. • D arm Contains dihydrouridine (D) Contributes to folding • TψC arm Contains pseudouridine (ψ)―has bonding between base and ribose Helps in folding Structure and characteristic features of tRNA
- 57. 3D structure of yeast tRNA by X-ray Diffraction
- 58. • When different codons specify one amino acid, the difference lies at the third position of the codon (5’ end of tRNA anticodon) • Wobble or flexibility at this site allows the tRNA anti-codon to read multiple codon sequences • Minimum of 32 tRNAs are required to translate all 61 codons (31 for amino acids and 1 for initiation with fMet) Wobble base-pairing in anticodon
- 59. “Wobble” pairings in tRNA with mRNA can occur in the third base • The third base of a codon can form non-canonical base pairs with its complement (anticodon) in tRNA • Some tRNAs contain Inosinate (I), which can H-bond with U,C, and A • These H-bonds are weaker and were named by Crick as “wobble” base pairs • Example: In yeast, CGA, GCU, and CGC all bind to tRNAArg, which has the anticodon 3’-GCI-5’ • Although sequences are usually written 5’🡪3’, the anticodon here is written 3’🡪5’ to illustrate its bonding to the mRNA codons
- 60. “Wobble” pairings in bacteria and eukaryotes
- 61. Anticodon containing inosinate (G or A) can pair with any of 3 codon sequences
- 62. Ribosome and polypeptide synthesis • Ribosome is a large protein synthesis machine • It finds the specific start site that sets the reading frame • As the ribosome moves along the mRNA, it translates codons to amino acids using tRNA • At the end, stop codon, it releases protein to cytoplasm and detach itself • 2 Subunits of ribosome • “Small” subunit involved in the recognition of the mRNA and tRNA sequences • “Large” subunit attaches the amino acid to form a polypeptide chain
- 63. 1) Activation of amino acids • tRNA is aminoacylated 2) Initiation of translation • mRNA and aminoacylated tRNA bind to ribosome 3) Elongation • Cycles of aminoacyl-tRNA binding and peptide bond formation…until a STOP codon is reached 4) Termination and ribosome recycling • mRNA and protein dissociate, ribosome recycled 5) Folding and post-translational processing • Catalyzed by a variety of enzymes Five stages of protein synthesis
- 64. Overview of protein synthesis
- 65. Additional reading – translation
- 66. Additional reading – mechanism of translation
- 67. Next Class – Analyzing and Sequencing Nucleic Acids • Copying DNA • Amplifying DNA • Probing DNA • Measuring DNA length • Sequencing DNA
- 68. Thank You