This document provides an overview of the central dogma of biology and DNA replication. It begins by defining the central dogma as the flow of genetic information from DNA to RNA to proteins. It then discusses the four requirements for DNA to be the genetic material and explains DNA replication through semi-conservative replication and starting at the origin. The basics of transcription and translation are also summarized, including the components and steps of each process.
This presentation deals with the ‘Central Dogma’ which is briefly the process by which the instructions in DNA are converted into a functional product. It was first proposed in 1958 by Francis Crick, discoverer of the structure of DNA.
The genetic material of a cell or an organism refers to those materials found in the nucleus, mitochondria and cytoplasm, which play a fundamental role in determining the structure and nature of cell substances, and capable of self-propagating and variation.
This presentation deals with the ‘Central Dogma’ which is briefly the process by which the instructions in DNA are converted into a functional product. It was first proposed in 1958 by Francis Crick, discoverer of the structure of DNA.
The genetic material of a cell or an organism refers to those materials found in the nucleus, mitochondria and cytoplasm, which play a fundamental role in determining the structure and nature of cell substances, and capable of self-propagating and variation.
it describes transcription with simple diagram and animation. its steps and inhibitors are described for both eukaryotes and prokaryotes. it will be easily understood by UG students . post transcriptional modification of all the RNA are also described with diagrams.
RNA- A polymer of ribonucleotides, is a single stranded structure. There are three major types of RNA- m RNA,t RNA and r RNA. Besides that there are small nuclear,micro RNAs, small interfering and heterogeneous RNAs. Each of them has a specific structure and performs a specific function.
it describes transcription with simple diagram and animation. its steps and inhibitors are described for both eukaryotes and prokaryotes. it will be easily understood by UG students . post transcriptional modification of all the RNA are also described with diagrams.
RNA- A polymer of ribonucleotides, is a single stranded structure. There are three major types of RNA- m RNA,t RNA and r RNA. Besides that there are small nuclear,micro RNAs, small interfering and heterogeneous RNAs. Each of them has a specific structure and performs a specific function.
Genetic Engineering and Biotechnology, a frontier discipline of modern science, has facilitated revolutionary developments in the fields of agriculture, industry, health and environment by genomic modification of animals, plants and microorganisms.
DNA replication is an important process which takes place in every organisms, be it prokaryotic or eukaryotic. The DNA replication process produces two identical copies of daughter DNA molecules using the existing DNA molecule as template. Each daughter DNA molecule inherits one strand from the parent cell and the other strand is newly synthesized. This is known as semiconservative mode of replication, demonstrated by Meselson and Stahl.
DNA is the genetic material that defines every cell. Before a cell duplicates and is divided into new daughter cells through either mitosis or meiosis, biomolecules and organelles must be copied to be distributed among the cells. DNA, found within the nucleus, must be replicated in order to ensure that each new cell receives the correct number of chromosomes. The process of DNA duplication is called DNA replication. Replication follows several steps that involve multiple proteins called replication enzymes and RNA. In eukaryotic cells, such as animal cells and plant cells, DNA replication occurs in the S phase of interphase during the cell cycle. The process of DNA replication is vital for cell growth, repair, and reproduction in organisms.
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Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
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2. INTRODUCTION
“The central dogma of molecular biology deals with the detailed
residue-by-residue transfer of sequential information. It states that
such information cannot be transferred back from protein to either
protein or nucleic acid.”
Francis Crick, 1958
3. • Protein information cannot flow
back to nucleic acids
• Fundamental framework to
understanding the transfer of
sequence information between
biopolymers
4. The central dogma of biology is that information
stored in DNA is transferred to RNA molecules during
transcription and to proteins during translation.
DNA RNA proteins
Genotyping Phenotyping
RNA DNA/RNA proteins
virus
5. FOUR REQUIREMENTS FOR DNA TO
BE GENETIC MATERIAL
Must carry information
• Cracking the genetic code
Must replicate
• DNA replication
Must allow for information to change
• Mutation
Must govern the expression of the phenotype
• Gene function
6. DNA REPLICATION
Process of duplication of the entire genome prior to cell
division
Biological significance
• extreme accuracy of DNA replication is necessary in
order to preserve the integrity of the genome in
successive generations
• In eukaryotes , replication only occurs during the S
phase of the cell cycle.
• Replication rate in eukaryotes is slower resulting in a
higher fidelity/accuracy of replication in eukaryotes
7. BASIC RULES OF REPLICATION
A. Semi-conservative
B. Starts at the ‘origin’
C. Synthesis always in the 5-3’ direction
D. Can be uni or bidirectional
E. Semi-discontinuous
F. RNA primers required
9. Semi-conservative
replication:
One strand of duplex
passed on unchanged to
each of the daughter
cells. This 'conserved'
strand acts as a template
for the synthesis of a
new, complementary
strand by the enzyme
DNA polymerase
10. HOW DO WE KNOW THAT DNA REPLICATION IS
SEMICONSERVATIVE?
MESELSON-STAHL EXPERIMENTS
11. B) STARTS AT ORIGIN
Initiator proteins identify specific base sequences on DNA
called sites of origin
Prokaryotes – single origin site E.g E.coli - oriC
Eukaryotes – multiple sites of origin (replicator)
E.g. yeast - ARS (autonomously replicating sequences)
Prokaryotes Eukaryotes
12. In what direction does DNA replication occur?
Where does energy for addition
of nucleotide come from?
What happens if a base
mismatch occurs?
C) Synthesis is ALWAYS in the 5’-3’ direction
13. Why does DNA replication only occur in the 5’ to 3’ direction?
Should be PPP here
14. D) UNI OR BIDIRECTIONAL
Replication forks move in one or opposite directions
15. E) SEMI-DISCONTINUOUS REPLICATION
Anti parallel strands replicated simultaneously
Leading strand synthesis continuously in 5’– 3’
Lagging strand synthesis in fragments in 5’-3’
18. Core proteins at the replication fork
Topoisomerases
Helicases
Primase
Single strand
binding proteins
DNA polymerase
Tethering protein
DNA ligase
- Prevents torsion by DNA breaks
- separates 2 strands
- RNA primer synthesis
- prevent reannealing
of single strands
- synthesis of new strand
- stabilises polymerase
- seals nick via phosphodiester linkage
19. THE MECHANISM OF DNA REPLICATION
Arthur Kornberg, a Nobel prize winner and other
biochemists deduced steps of replication
• Initiation
• Proteins bind to DNA and open up double helix
• Prepare DNA for complementary base pairing
• Elongation
• Proteins connect the correct sequences of
nucleotides into a continuous new strand of DNA
• Termination
• Proteins release the replication complex
21. 21
PROOFREADING NEW DNA
• DNA polymerase initially makes about 1 in
10,000 base pairing errors
• Enzymes proofread and correct these mistakes
• The new error rate for DNA that has been
proofread is 1 in 1 billion base pairing errors
22. 22
DNA DAMAGE & REPAIR
• Chemicals & ultraviolet radiation damage the
DNA in our body cells
• Cells must continuously repair DAMAGED
DNA
• Excision repair occurs when any of over 50
repair enzymes remove damaged parts of DNA
• DNA polymerase and DNA ligase replace and
bond the new nucleotides together
23. TRANSCRIPTION
• Process of copying DNA to RNA
• Differs from DNA synthesis in that only one strand of DNA,
the template strand, is used to make mRNA
• Does not need a primer to start
• Can involve multiple RNA polymerases
• Divided into 3 stages
• Initiation
• Elongation
• Termination
29. TYPES OF RNA MOLECULES
• Messenger RNAs (mRNAs)—intermediates that
carry genetic information from DNA to the
ribosomes.
• Transfer RNAs (tRNAs)—adaptors between
amino acids and the codons in mRNA.
• Ribosomal RNAs (rRNAs)—structural and
catalytic components of ribosomes.
31. TRANSLATION: INITIATION
• Ribosome small subunit binds to mRNA
• Charged tRNA anticodon forms base pairs with the mRNA codon
• Small subunit interacts with initiation factors and special initiator
tRNA that is charged with methionine
• mRNA-small subunit-tRNA complex recruits the large subunit
• Eukaryotic and prokaryotic initiation differ slightly
32. TRANSLATION: INITIATION
•The large subunit of the ribosome contains three binding sites
•Amino acyl (A site)
•Peptidyl (P site)
•Exit (E site)
•At initiation,
•The tRNAfMet occupies the P site
•A second, charged tRNA complementary to the next codon
binds the A site.
33. TRANSLATION: ELONGATION
• Elongation
• Ribosome translocates by three bases after peptide bond formed
• New charged tRNA aligns in the A site
• Peptide bond between amino acids in A and P sites is formed
• Ribosome translocates by three more bases
• The uncharged tRNA in the A site is moved to the E site.
34. TRANSLATION: ELONGATION
• EF-Tu recruits charged tRNA to A site. Requires hydrolysis of
GTP
• Peptidyl transferase catalyzes peptide bond formation (bond
between aa and tRNA in the P site converted to peptide bond
between the two amino acids)
• Peptide bond formation requires RNA and may be a ribozyme-
catalyzed reaction
35. TRANSLATION: TERMINATION
• Termination
• Elongation proceeds until STOP codon reached
UAA, UAG, UGA
• No tRNA normally exists that can form base pairing with a STOP
codon; recognized by a release factor
• tRNA charged with last amino acid will remain at P site
• Release factors cleave the amino acid from the tRNA
• Ribosome subunits dissociate from each other
• Review the animation of translation
36.
37. REFRENCES:-
• Life sciences, fundamentals and practices-2,pranav
kumar and usha mina,5th edition,2016.
• Slideshare.com
• http://www.thelifewire.com