More Related Content
Similar to structure of dna and transcription (20)
structure of dna and transcription
- 1. INTRODUCTION TO GENETICS SUBMITTED BY: ANUPAM PRAHLAD FIRST M.PHARM
- 2. GENETICS – The branch of biology that deals with heredity, especially the mechanisms of hereditary transmission and the variation of inherited characteristics among similar or related organisms
- 3. GENE • Biological unit of heredity • Gene hold the information to build and maintain their cells and pass genetic traits to offsprings • In cells, a gene is portion of DNA
- 4. Gene (DNA) RNA formation Protein formation Cell structure Cell enzymes cell function
- 5. CHROMOSOMES • They are the rod-shaped, filamentous bodies present in the nucleus, which become visible during cell division. • They are the carriers of the gene or unit of heredity • Chromatin: DNA, RNA & proteins that make up chromosme • Chromatids: one of the two identical parts of the chromosome • Centromere: the point where two chromatids attach
- 6. • 46 chromosomes : 22 pairs Autosomes and 1 pair Sex chromosomes.
- 7. DNA • Deoxyribonucleic acid (DNA) is a molecule that carries the genetic instructions used in the growth, development, functioning and reproduction of all known living organisms and many viruses. • DNA and RNA are nucleic acids. • They consist of four deoxynucleotides • DNA has three main components 1. deoxyribose (a pentose sugar) 2. base (there are four different ones) 3. phosphate
- 8. • Bases are divided into two groups 1.Pyrimidines 2. Purines PURINE BASES • Made of a 6 member ring, fused to a 5 membered ring. • They comprises of adenine and guanine • Adenine is 6-amino purine and guanine is 2-amino,6-oxypurine
- 9. PYRIMIDINE BASES • Made up of a 6member ring. • The pyrimidine bases are cytosine and thymine. • Cytosine is present in both DNA and RNA • Thymine is present only in DNA. uracil is present in RNA
- 10. NUCLEOTIDES • They are phosphate esters of nucleosides • Base + sugar +phosphate = nucleotide • The esterification occurs at the fifth and third hydroxyl group of pentose sugar. • Nucleotides are linked by covalent bonds called PHOSPHODIESTER LINKAGES
- 11. STRUCTURE OF DNA • DNA is apolymer of deoxribo nucleotides . • It s made of monomeric units mainly dAMP, dGMP, dDCMP, dTMP • Made of two strands of nucleotides that are joined together by hydrogen bonding • Hydrogen bonding occurs as a result of complimentary base pairing. • Adenine and thymine pair up • Cytosine and guanine pair up • Each pair is connected through hydrogen bonding • Hydrogen bonding always occurs between one pyrimidine and one purine
- 12. • The monomeric deoxynucleotides in DNA are held together by 3’-5’phosphodiester bridges • DNA structure is often represented in a short hand form.
- 13. CHARGAFF’S RULE OF DNA DECOMPOSITION • Erwin Chargaff in late 1940’s quantitatively analysed the DNA hydrolysates from different species. • He observed that in all species DNA had equal number of adenine and thymidine residues ( A=T) and equal number of guanine and cytosine residues(G=C) • This is known as Chargaff’s rule of molar equivalence between the purines and pyrimidenes
- 14. WATSON AND CRICK MODEL OF DNA • Watson and Crick used the empirical data of Franklin, Wilkin, and Chargaff to come up with a model of the DNA structure. • It was an important finding to the field of molecular biology and genetics. • They published a 900 words paper and Franklin and Wilkin also published on the same issue of Nature.
- 15. 1) DNA is a double helix. • It is aright handed double helix. 2) Two polynucleotides chains It consist of 2 polydeoxyribonucleotide chain twisted around each other on a common axis
- 16. 3.The two chains wind around right handedly - right handed double helix.
- 17. 4. Two chains are in an anti-parallel orientation. (one strand 5’ – 3’ orientation and the other 3’ – 5’).
- 18. 5. Each strand of DNA has a hydrophilic deoxy ribose phosphate backbone( 3’-5’ phosphodiester bonds) on the outside of molecule while the hydrobhobic bases are stacked inside the core • Sugar-phosphate backbone is located on the outside of the helix
- 19. 6. The bases are stacked flat and perpendicular to the two polynucleotides are bonded together via hydrogen bonds on the inside of the helix. The bases are on top of each other following the twist of the helix.
- 20. 7. The 2 strands are held together by hydrogen bonds formed by complementary base pairs.the A-T base air has 2 hdrogen bonds • The G-C has 3 hydrogen bonds. The G=C is stronger b about 50% than A=T
- 21. 8. The complementary base pairing in DNA helix proves chargaff’s rule. 9. Bases of the two polynucleotide chains are base- pairing to maintain similar diameter of the double helix.
- 22. 10. The sequence of one chain (strand) is enough to predict the complementary one in the other orientation 11. there are two grooves found in DNA molecule namely Major groove and Minor groove – The width of DNA molecule is 20 Å. – The strand completes a turn every 34 Å along its length. – There are ten nucleotides per turn. – The inter nucleotide distance 3.4 Å.
- 23. CONFIRMATIONS OF DNA DOUBLE HELIX • Variation in the confirmation of the nucleotides of DNA is associated with the confirmational variants of DNA • The double helical structure of DNA exist in atleast 6 different forms, A-E and Z. • The B form of DNA double helix described by Watson and Crick is the most predominant form under physiological conditions • Each turn of the B form has 10 base pairing spanning a distance of 3.4nm. • The width is about 2 nm.
- 24. • The A form is also a right handed helix • It contains 11 base Pair per turning • There is a tilting of the base air from central axis • The Z form is left handed helix and contains 12 base pairs per turn • The polynucleotide strands of DNA move in a somewhat zig zag fashion, hence the name Z-DNA
- 25. COMPARISON OF SRUCTURAL FEATURES OF DIFFERENT CONFIRMATIONS OF DNA HELIX FEATURE B-DNA A-DNA Z-DNA Helix type Right handed Right handed Left handed Helical diametre 2.37 2.55 1.84 Distance per each complete turn(nm) 3.4 3.2 4.5 Rise per base pair .34 .29 .37 No:of base pairs per complete turn 10 11 12 Base pair tilt +19 -1.2 -9 Helix axis rotation Major groove Through base pairs Minor groove
- 26. OTHER TYPES OF DNA STRUCTURE 1.Bent DNA • In general ,adenine base containing DNA tracts are rigid and straight • Bent confirmations of DNA occurs when A-tracts are replaced by other bases or a collapse of the helix into the minor groove of A tract • Bending has also been reported due to photochemical damage or mispairing of bases • Certain antitumor drugs (cisplatin) produce bent structures in DNA
- 27. 2.TRIPLE STRANDED DNA – Occur due to additional hdrogen bond between the bases – Thus a thymine can selectively form 2 Hoogsten hydrogen bonds to the adenine of A-T pair to form T-A-T. – Likewise, cytosine can form C-G-C. – They are less stable than double helices due to the fact that the three negatively charged backbone strands in triple helix results in increased electrostatic repulsion
- 28. • Polynucleotide with very high contents of guanine can form a novel tetrameric structure called G-quartlets • These structures are planar and are connected by Hoogsteen bonds • Antiparallel four stranded DNA structures referred to as G- tetralexes have also been reported • G tetralexes have been implicated in the recombination f immunoglobulin genes and in dimerisation of double stranded genomic RNA of HIV.
- 30. DNA REPLICATION
- 31. • When the cell divides , the daughter cell receive an identical copy of genetic information from the parent cell • Replication is a process in which DNA copies itself to produce identical daughter molecule of DNA • It is a complex process that occurs in all living organism and copies their exact DNA. • It is the basis for biological inheritance • Replication is carried out with high tidelity which is essential for the survival of species • Delbruck suggested that Watson-Crick model of DNA could theoritically be replicated by 3 modes -conservative -semi conservative -dispersive
- 32. • MESELSON and STAHL in 1958 proved that DNA replication is semi conservative in vivo • In the daughter cell, one strand is derived from mother cell while the other strand is newly synthesised • This is called semi conservative type of DNA replication • Each strand serve as a template over which a new complementary strand is produced
- 33. • Parenteral strands are not degraded • Base pairing allows each strand to serve as a template strand for a new strand • New duplex is ½ parent template and ½ new DNA
- 34. STEPS IN DNA REPLICATION 1) Identification of origin of replication 2) Unwinding of DNA to provide a template strand 3) Formation of replication fork . 4) Direction of DNA replication 5) Synthesis of RNA primer 6) chain elongation 7) Excision of RNA primer and their replacement by DNA 8) DNA ligase action 9) Termination
- 35. INITIATION • DNA replication initiate from specific sequences of Origin of replication (ORI) called Replisomes. • The origin of replication in bacteria is called ori whereas in higher organisms known as replicators • This area is recognized by specific proteins called origin recognition complex • Eukaryotic cells have multiple replication sites. • To initiate replication process, multiple replicative proteins must assemble on these replication sites. • It leads to formation of Pre-replication complex (pre-RC).
- 36. Pre-replication complex has steps : 1.association of Origin recognizing complex (ORC) with replication origin. 2. binding of Cdc6 protein to ORC 3. binding of Cdt1 and minichromosome maintenance protein. • This replicative complex assembly occurs during G1 phase prior to S phase. • During the transition between G1 phase to S phase, CDK proteins and DDK proteins get attached to the Pre- replication complex. • It transforms the Pre-replication complex into active replication fork.
- 38. REPLICATION FORK • As the two strands unwind and separate, they form a “Y shaped” where active synthesis occurs. This region is called the replication fork. • DNA helicase unwinds the double helix. • The replication fork moves at the rate of 1000 nucleotides per second. • SSB protein helps to keep the strand separated. • As the two strands of the double helix are separated, a problem is encountered, namely, super-coiling in the region of DNA ahead of the replication fork.
- 39. • The accumulating positive supercoils interfere with further unwinding of the double helix • To solve the problem of super-coiling, there is a group of enzymes called DNA topoisomerases, which are responsible for removing supercoils in the helix. • These enzymes reversibly cut one strand of the double helix. • They have both nuclease (strand-cutting) and ligase (strand- resealing) activities.
- 40. DIRECTION OF REPLICATION • The DNA polymerases responsible for replication are only able to “read” the parental nucleotide sequences in the 3′→5′ direction, and they synthesize the new DNA strands only in the 5′→3′ (anti- parallel) direction. Leading strand • The leading strand is the strand of nascent DNA which is being synthesized in the same direction as the growing replication fork. • A polymerase "reads" the leading strand template and adds complementary nucleotides to the nascent leading strand on a continuous basis. Lagging strand: This strand is extended away from the replication fork and synthesized discontinuously in small fragments known as Okazaki fragments, each requiring a primer to start the synthesis.
- 41. RNA PRIMER • DNA polymerases cannot initiate synthesis of a complementary strand of DNA on a totally single-stranded template. Rather, they require an RNA primer, with a free hydroxyl group on the 3′-end of the RNA strand. • A specific RNA polymerase, called Primase (DnaG), synthesizes the short stretches of RNA (approximately ten nucleotides long) that are complementary and anti- parallel to the DNA template. • These short RNA Primer are constantly being synthesized at the replication fork on the lagging strand, but only one RNA sequence at the origin of replication is required on the leading strand.
- 42. CHAIN ELONGATION • DNA polymerases elongate a new DNA strand by adding deoxy- ribonucleotides, one at a time, to the 3′-end of the growing chain. • DNA chain elongation is catalyzed by DNA polymerase III. • The new strand grows in the 5′→3′ direction, anti- parallel to the parental strand . • Pyrophosphate (PPi) is released when each new deoxynucleoside monophosphate is added to the growing chain. • This newly added nucleotide would now polymerase with one another forming the next phosphodiester bond.
- 43. EXCISION OF RNA PRIMERS AND THEIR REPLACEMENT BY DNA • DNA POL I removes the RNA primer and fills the gap between Okazaki fragments. DNA LIGASE ACTION • The final phosphodiester linkage between the 5′-phosphate group and the 3′-hydroxyl group on the chain is catalyzed by DNA ligase. TERMINATION • Termination of DNA replication in E. coli is mediated by binding of the protein, TUS (Terminus Utilization Substance) to replication termination sites (Ter sites) on the DNA, stopping the movement of DNA polymerase.
- 44. TRANSCRIPTIO N
- 45. – Transcription is the first step of gene expression, in which a particular segment of DNA is copied into RNA (especially mRNA) by the enzyme RNA polymerase. Transcription proceeds in the following general steps: – RNA polymerase, together with one or more general transcription factors, binds to promoter DNA. – RNA polymerase creates a transcription bubble, which separates the two strands of the DNA helix. This is done by breaking the hydrogen bonds between complementary DNA nucleotides. – RNA polymerase adds RNA nucleotides (which are complementary to the nucleotides of one DNA strand).
- 46. – RNA sugar-phosphate backbone forms with assistance from RNA polymerase to form an RNA strand. – Hydrogen bonds of the RNA–DNA helix break, freeing the newly synthesized RNA strand. – If the cell has a nucleus, the RNA may be further processed. This may include polyadenylation, capping, and splicing. – The RNA may remain in the nucleus or exit to the cytoplasm through the nuclear pore complex.
- 47. TEMPLATE STRAND – The strand that is transcribed or copied into an RNA molecule is referred to as the template strand of the DNA. – The other DNA strand, the non-template strand, is frequently referred to as the coding strand of that gene. Biochemistry For Medics- Lecture Notes 6 – The information in the template strand is read out in the 3' to 5' direction – The sequence of ribonucleotides in the RNA molecule is complementary to the sequence of deoxy ribonucleotides in template strand of the double-stranded DNA molecule – In the coding strand (complementary strand) the sequence is same as that of the sequence of nucleotides in the primary transcript.
- 48. TRANSCRIPTION UNIT – A transcription unit is defined as that region of DNA that includes the signals for transcription initiation, elongation, and termination. – DNA-dependent RNA polymerase is the enzyme responsible for the polymerization of ribonucleotides into a sequence complementary to the template strand of the gene. – The enzyme attaches at a specific site—the promoter—on the template strand. – This is followed by initiation of RNA synthesis at the starting point, and the process continues until a termination sequence is reached.
- 50. PRIMARY TRANSCRIPT – The RNA product, which is synthesized in the 5' to 3' direction, is the primary transcript. – In prokaryotes, this can represent the product of several contiguous genes – In mammalian cells, it usually represents the product of a single gene – The 5' terminals of the primary RNA transcript and the mature cytoplasmic RNA are identical. – The starting point of transcription corresponds to the 5' nucleotide of the mRNA. – This is designated position +1, as is the corresponding nucleotide in the DNA
- 51. – The numbers increase as the sequence proceeds downstream. – The nucleotide in the promoter adjacent to the transcription initiation site is designated -1, – These negative numbers increase as the sequence proceeds upstream, away from the initiation site
- 52. DNA-Dependent RNA Polymerase – The DNA-dependent RNA polymerase (RNAP)is the complex consisting of -two identical α subunits -similar but not identical β and β ' subunits, -ω subunit -A sigma subunit (σ) -Beta is thought to be the catalytic subunit RNAP, a metalloenzyme, also contains two zinc molecules. The core RNA polymerase associates with a specific protein factor (the sigma σ factor) that helps the core enzyme recognize and bind to the specific deoxynucleotide sequence of the promoter region to form the preinitiation complex (PIC)
- 53. • Mammalian cells possess three distinct nuclear DNA-Dependent RNA Polymerases • RNA polymerase I is for the synthesis of r RNA • RNA polymerase II is for the synthesis of m RNA and miRNA • RNA polymerase III is for the synthesis of tRNA/5S rRNA, snRNA
- 54. STEPS OF RNA SYNTHESIS • Initiation phase: RNA-pol recognizes the promoter and starts the transcription. • Elongation phase: the RNA strand is continuously growing. • Termination phase: the RNA-pol stops synthesis and the nascent RNA is separated from the DNA template.
- 55. i) Initiation of Transcription – Initiation of transcription involves the binding of the RNA polymerase holoenzyme to the promoter region on the DNA to form a preinitiation complex, or PIC – Characteristic "Consensus" nucleotide sequence of the prokaryotic promoter region are highly conserved Pribnow box – This is a stretch of 6 nucleotides ( 5'- TATAAT-3') centred about 8-10 nucleotides to the left of the transcription start site. -35 Sequence – A second consensus nucleotide sequence ( 5'- TTGACA-3'), is centred about 35 bases to the left of the transcription start site.
- 56. – Binding of RNA-polymerase (RNAP) to the promoter region is followed by a conformational change of the RNAP, and the first nucleotide (almost always a purine) then associates with the initiation site on the subunit of the enzyme. – In the presence of the appropriate nucleotide, RNAP catalyzes the formation of a phosphodiester bond, and the nascent chain is now attached to the polymerization site on the subunit of RNAP. – In both prokaryotes and eukaryotes, a purine ribonucleotide is usually the first to be polymerized into the RNA molecule. – After 10–20 nucleotides have been polymerized, RNAP undergoes a second conformational change leading to promoter clearance. – Once this transition occurs, RNAP physically moves away from the promoter, transcribing down the transcription unit, leading to the next phase of the process, elongation.
- 57. Elongation step of Transcription – As the elongation complex containing the core RNA polymerase progresses along the DNA molecule, DNA unwinding must occur in order to provide access for the appropriate base pairing to the nucleotides of the template strand. – The extent of this transcription bubble (i.e., DNA unwinding) is constant throughout and is about 20 base pairs per polymerase molecule – RNA polymerase has associated with it an "unwindase" activity that opens the DNA helix. – Topo isomerase both precedes and follows the progressing RNAP to prevent the formation of super helical complexes. – Base pairing rule is followed during the incorporation
- 59. Termination of transcription – Termination of the synthesis of the RNA molecule in bacteria is of two types a) Rho (ρ) dependent termination b)Rho (ρ) independent termination Rho (ρ) dependent termination The termination process is signalled by a sequence in the template strand of the DNA molecule—a signal that is recognized by a termination protein, the rho (ρ) factor. Rho is an ATP-dependent RNA-stimulated helicase that disrupts the nascent RNA-DNA complex
- 61. Rho independent termination This process requires the presence of intrachain self complementary sequences in the newly formed primary transcript so that it can acquire a stable hair pin turn that slows down the progress of the RNA polymerase and causes it to pause temporarily. Near the stem of the hairpin, a sequence occurs that is rich in G and C. This stabilizes the secondary structure of the hair pin.
- 62. – Beyond the hair pin, the RNA transcript contains a strings of Us, the bonding of Us to the corresponding As is weak. – This facilitates the dissociation of the primary transcript from DNA. – After termination of synthesis of the RNA molecule, the enzyme separates from the DNA template. – With the assistance of another factor, the core enzyme then recognizes a promoter at which the synthesis of a new RNA molecule commences
- 63. RNA SPLICING In eukaryotes RNA transcripts have long non-coding stretches of nucleotides -these regions will not be translated . • The non-coding sections are dispersed between coding sections • Introns-non-coding sections of nucleic acid found between coding regions • Exons -coding regions of nucleic acids (eventually these are expressed as amino acids) • RNA polymerase transcribes introns and exons-this is pre- mRNA . • Pre-mRNA never leaves the cell’s nucleus. • The introns are excised and exons are joined together to form mRNA
- 65. THANK YOU