Also it is exposed to many chemical, physical, and
biological agents which enters the cell from
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environment.All such agents inflict a variety of damages on DNA
molecule.
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Maintenance of the integrity of DNA is vital for the
survival and continuation of species, Hence DNA repair
mechanisms to take care of these damages.
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CAUSES OF DNA DAMAGE.Replication error----
normally errors are repaired by
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the proof reading mechanisms of DNA replication andare not transmitted. But when they are increases or are
not repaired properly, they can give rise to disease.
CHEMICALS.
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A wide variety of chemicals are present inenvironment to which all of us are exposed .
Examples are ?
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Insecticide and pesticides.Pollutants including gases.
Oxidising agents, alkylating agents.
Industrials wastes
Food adulteration and preservative.
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All the chemicals causes modification of bases,Bulky adduct formation between bases, alteration
of bases by deamination, etc and lead to DNA
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damage.
DEAMINATION OF BASES.
Cytosine and adenine undergo deamination
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spontaneous or chemically induced to form uracil and
hypoxanthine . The original sequence is thus changed.
Thymus Dimers by UV Light.
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UV light radiations induce the condensation of adjacent thyminebases in DNA strand forming thymine- thymine dimers.
CHAIN BREAKS BY IONIZING RADIATION.
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X-rays and
gamma rays can cause DNA strand in the backbone in single
strand or in both the strands.
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DAMAGE BY OXYGEN RADICALS.
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ADDUCT FORMATION.MECHANISM OF DNA REPAIR.
BASE EXCISION REPAIR- modified and altered bases
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are repaired by base excision repair mechanism.NUCLEOTIDE EXICION REPAIR- The repair mechanism
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involves called Excinuclease. Exinuclease cuts nucleotide,The gap is filed by DNA polymerase.
DIRECT THYMINE DIMER REPAIR- DNA photolyase
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catalyse the removal of bonds forming thymine dimers
and converting them to back to normal in prokaryotes.
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MISMATCH REPAIR ? specific endonucleases identify thewrong base in newly synthesized strand.
TRANSCRIPTION.
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Transcription-cellular process in which RNA synthesized
using DNA template known transcription.
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ROLE OF DNA --- only one strand used.-Template strand/non ?coding strand.
-non template strand/coding strand.
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Transcription.
ROLES OF DNA AND RNA.
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DNA is the MASTER PLAN.RNA is the BLUPRINT of the Master plan.
Transcription---
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process in which RNA is synthesized fromDNA.
DNA express through RNA.
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Synthesis of RNA occurs in 5'-3' direction.Nucleotide ATP, GTP,CTP,UTP ARE NECESSORY.
Enzyme and factors.
Eukaryotes
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prokaryotes
RNA POLYMERASE
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RNA POLYMERASERNA polymerase I
Only one type of RNA polymerase
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RNA polymerase II
Sigma factor.
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RNA polymerase IIIDIFFERENCE BETWEEN REPLICATION
ANDTRANSCRIPTION
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REPLICATION
TRANSCRIPTON
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TEMPLATEBOTH STRAND
SINGLE STRAND
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PRIMER
YES
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NOENZYME
DNA POLYMERASE
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RNA POLYMERASE
PRODUCT
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Ds DNAssRNA
BASE PAIR
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A -T, G-C,
A-U, T-A, G-C
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PROOF READINGYES
NO
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DNA dependent RNA polymerase.RNA polymerase main enzyme responsible for
transcription.
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Five core unit and sixth sigma factor to make
holoenzyme.
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Beta subunit has polymerisation activity.RNAP reads the template strand in 3'-5' direction
and synthesize new RNA in 5'-3' direction and does
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not require a RNA primer.RNA polymearase.
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RNA polymerase.
STEPS OF TRANSCRIPTION.
Initiation.
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Elongation.Termination.
Initiation----
RNAP binds to a region of gene DNA called
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promoter with the help of sigma factor.
RNA polymerase binds to DNA promoter.
Initiation require special DNA sequence.
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Recognise DNA strand.
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Transcription.INITIATION OF TRANSCRIPTION.
RNAP binding?
initiation begins with the binding of
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RNAP to the promoters of the DNA to form pre
initiation complex.
RNAP has an associated unbinding activity.
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Binding is followed by a change in RNAP conformation.
This places Beta subunit of RNAP on the first
nucleotide of the initiation site.
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The RNAP then catalyzes polymerisation of the first
nucleotide with the second nucleotide according to the
template strand sequence.
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Promoter region.
Elongation.
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The RNA polymerase core enzyme progresses along DNAmolecule to continue elongation.
The elongation continues till the termination signals are
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encountered.
TERMINATION--
Two types of termination signals are
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present.
Rho- factor dependent signals...............
Rho- factor independent signals.............
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TerminationEukaryotes
Prokaryotes
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Polyadenylation signal(AAUAAA)
Rho-factor dependent.......
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transcribed.Reaches to -35 bp downstream
Rho- factor independent......
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RNA transcript released.INITIATION.
INITIATION.
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The process of RNA synthesis inbacteria begins with the binding of RNA polymerase
molecule on the DNA.. RNAP recognises the
transcription start site on the template strand of DNA
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with the help of certain specific region on the DNAknown as promoter element.
PE region identified in bacteria---
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1.Pribnow box2.The `-35' sequence
ELONGATION.
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ELONGATION---The elongation of the RNA moleculeoccurs from the 5' to its 3' end, antiparallel to the
template strand. RNAP assembles ribonucleotides in a
complementary sequence to the tepmlate strand by
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watson ? crick base pairing rule.TERMINATION.
TERMINATION--
Two type of such signals are are
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identified in prokaryotes.
1.Rho factor? it is a protein that binds either to
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the growing RNA or template strand of DNA anddissociate the the RNAP from DNA, thus
terminating the synthesis of RNA. The released
RNAP associates with the sigma factor( which was
released earlier during initiation) and is recycled.
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TRANSCIRIPTION IN EUKARYOTES.Much more complicated.
Three different RNA polymerase.
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Required many transcription factor protein.
Transcription initiation needs promoter and upstream
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regulatory regions.Enhancer /silencer --are DNA sequences that regulate
the rate of initiation of transcription by RNA
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polymerase II.PROMOTERS.
TRANSCRIPTION IN EUKARYOTES.
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Eukaryotes transcription is more complex compound to
that of prokaryotes. There exist three distinct RNA
polymerases transcribing different type of RNAs
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compared to the single RNAP in prokaryote.RNAP I ----for r RNA.
RNAP II ---for m RNA.
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RNAP III--- for t RNA and small r RNA.
INITIATION---
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Transcription starts with the RNAPrecognising the promoter site.
TATA box---similar to the pribnow box in prokaryotes a
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highly conserved nucleotide base sequence ( TATAAA)
occurs about -25 bp upstream to the TIS in eukaryotes. This
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sequences is also called as Goldberg- Hogness box.ENHANCER SEQUENCE-- increases the rate of
transcription.
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SILENCER SEQUENCE--- Decreases the rate of
transcription.
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HORMONE RESPONSE ELEMENT-- are the sites used bythe hormones to regulate the transcription.
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INITIATION COMPLEX.CAAT box--Further upstream to the TIS (-70 bp ),
there occurs another sequence (GGCCAATCT)
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known as CAAT box.PRE-INITIATION COMPLEX ?
RNA polymerase of
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eukaryotes (e.g RNAP II ) can not recognise TATA and
CAAT boxes directly. A group of protein factor play a
role in recognising the promoter regions of DNA and
called as transcription factor. TF bind to the promoter
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on DNA sequentially along with the enzyme to form apre-initiation complex.
TRANSCRIPTION IN EUKARYOTES.
ELONGATION--
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Elongation of the nascent m RNA occurs
similar to that of prokaryotes.
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TERMINATION? once transcription is completed , it is
terminated and RNAP-II is dephosphorylated.
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POST-TRANSCRIPTIONALMODIFICATIONS.
The mRNA in prokaryotes is fully functional as soon as it is
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synthesised. on the contrary , eukaryotic RNA produced bytranscription as primary transcript are non-functional. They
undergo extensive structural alteration to produce the
mature functional molecule.
Messenger RNA.
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The primary transcript of mRNA synthesised by RNA
polymerase II in eukaryotes is often called
heterogeneous nuclear RNA. hn RNA is subjected to
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extensive processing in the nucleus to produce maturemRNA, which then enters the cytoplasm to take part in
protein synthesis.
Messenger RNA processing.
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The cap-The 5' terminal of mRNA is
crowned with a 7-methylguanosine cap.
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The tail-The 3' end of mRNA is attached by a number of
adenylate residues .This is known as poly-A tail.
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Splicing-
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During processing introns are removed and exons are
spliced together to form mature RNA.
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MICRO RNAs(miRNA) AND SMALL INTERFERINGRNA(siRNA).
Micro RNA and small interfering RNA are a class of small RNA involved
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in gene silencing.They form complimentary hybrid with the target DNA and lead to either
its degradation or inhibition of translation.
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Micro RNA- Long single stranded precursor RNAs.
INSIDE the nucleus, primary miRNA transcript is
first processed by a nuclease called DROSHA to produce a smaller
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double stranded hairpin loop like pre-miRNA.
This is exported out of the nucleus to cytoplasm and trimmed by
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nuclease enzyme DICER to produce double stranded miRNA duplex.Duplex miRNA unbound and one of the strand is selected, The selected
miRNA is loaded into the RNA induced silencing complex(RISC) to form
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a mature functional mi RNA.
It is used to silence silence target mRNA-------TRANSLATIONAL ARREST.
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