Download THE CENTRAL DOGMA OF MOLECULAR BIOLOGY Jony Mallik B

THE CENTRAL DOGMA OF MOLECULAR BIOLOGY
Jony Mallik
B. Pharmacy; M. Pharmacy
E-mail: [email protected]
INTRODUCTORY PRINCIPLE
Biomolecules are the molecules that are synthesized within living organism and
perform different functions in that organism in terms of separate metabolic &
biosynthetic purposes. The biomolecules could be a coarse one like- polypeptides,
polysaccharides, lipids or any other macromolecules. Protein is the only biomolecule
which is synthesized depending on body individual need & participate in different
biologic signaling system like plasma protein, membrane protein, receptor protein,
enzyme protein system. Proteins are the essential tools for proper growth & repair of
muscle. Some proteins, people may easily get from food stuff but, some are very
authentic & body usually biosynthesized that types of protein.
 In vivo protein synthesis and its phenomenon is the central dogma of molecular
biology.
 Central dogma of molecular biology is the explanation of two different steps
required in synthesizing a protein.
 Central dogma deals with the details of genetic information (DNA)
transformation to m RNA & then to a protein.
 The two vulnerable steps of protein synthesis are Transcription
 Translation
Deoxyribonucleic acid (DNA)
DNA is the molecule of life & a very essential nucleic acid located at the chromosome of
cellular nucleus. All the genetic information is stored in DNA molecule & transformed
into mRNA (Transcription) then to a protein (Translation), that is why DNA is
sometimes said as “The Reserve Bank of Genetic Information”.
 In DNA there are four bases: adenine (A), guanine (G), thymine (T) and cytosine
(C). Adenine and guanine are purines; thymine and cytosine are pyrimidines.
 A nucleoside is a pyrimidine or purine base covalently bonded to a sugar. In
DNA, the sugar is deoxyribose and so this is a deoxynucleoside. There are four
types
of
deoxynucleoside
in
DNA;
deoxyadenosine,
deoxyguanosine,
deoxythymidine and deoxycytidine.
 A nucleotide is base + sugar + phosphate covalently bonded together. In DNA,
where the sugar is deoxyribose, this unit is a deoxynucleotide.
 In DNA the nucleotides are covalently joined together by 3’5’phosphodiester
bonds to form a repetitive sugar–phosphate chain which is the backbone to
which the bases are attached.
 The DNA sequence is the sequence of A, C, G and T along the DNA molecule
which carries the genetic information.
 In a DNA double helix, the two strands of DNA are wound round each other
with the bases on the inside and the sugar–phosphate backbones on the outside.
The two DNA chains are held together by hydrogen bonds between pairs of
bases; adenine (A) always pairs with thymine (T) and guanine (G) always pairs
with cytosine (C).
DNA Replication
DNA replication is the process of the genesis of two identical replica of the parent DNA
molecule under the agency of some sorts of enzyme. Newly formed molecule contains
same genetic information that the parent molecule may have. Replication process
involve numerous complicated task. All the new molecule further participate in
transcription process.
Enzymes & Protein that play a key role in DNA replication process are
DNA polymerase I & III

DNA primase

DNA gyrase & nuclease

DNA helicase

DNA ligase

Single stranded DNA binding protein (SSB)
DNA Replication models
The process of DNA Replication was hiding many secrets. One of the most important
was how the two daughter strands are created. As we have noticed in previous chapters
of our site the DNA is a complex of two chains! In order the hereditary phenomenon to
be explained, these strands should be accurately copied and transmitted from the
parental cell to the daughter ones. These are three possible models that describe the
accurate creation of the daughter chains:
Semiconservative Replication According to this model, DNA Replication would create
two molecules. Each of them would be a complex of an old (parental and a daughter
strand).
Conservative Replication According to this model, the DNA Replication process would
create a brand new DNA double helix made of two daughter strands while the parental
chains would stay together.
Dispersive Replication According to this model the Replication Process would create
two DNA double-chains, each of them with parts of both parent and daughter
molecules.
Replication Process
The overall DNA replication process is very complicated job and involves a set of
proteins and enzymes that collectively assemble nucleotides in the predetermined
sequence. A particular replication process is consists of following distinct steps to form
two identical replica.
INITIATION
► The site from which the replication starts are called Replication origin or Origin of
replication. In order for DNA replication to begin, the double stranded DNA helix must
open, for that both of the helicase & SSB protein bind to that region to unwind the helix
& stabilize the DNA into two strand.
► The open portion of parent DNA are referred as “ Replication fork”, which is
asymmetrical as the two single strands run in a anti-parallel direction.
PRIMER SYNTHESIS
► The synthesis of a new, complementary strand of DNA using the existing strand as a
template is brought about by enzymes known as DNA polymerases. In addition to
replication they also play an important role in DNA repair and recombination.
► One of the most important steps of DNA Replication is the binding of RNA Primase
in the initiation point of the 3'-5' parent chain. RNA Primase can attract RNA
nucleotides which bind to the DNA nucleotides of the 3'-5' strand due to the hydrogen
bonds between the bases. RNA nucleotides are the primers (starters) for the binding of
DNA nucleotides.
SYNTHESIS OF LEADING STRAND
► Then DNA polymerase III play its role in initiating the leading strand which required
few steps & therefore is synthesized quickest.
► DNA polymerase III (DNA pol III) recognizes the 3' OH end of the RNA primer, and
adds new complementary nucleotides. As the replication fork progresses, new
nucleotides are added in a continuous manner, thus generating the new strand.
SYNTHESIS OF LAGGING STRAND
► DNA is synthesized in a discontinuous manner by generating a series small
fragments of new DNA in the 5' → 3' direction. These fragments are called Okazaki
fragments, which are later joined to form a continuous chain of nucleotides. This strand
is known as the lagging strand since the process of DNA synthesis on this strand
proceeds at a lower rate.
LIGATION & TERMINATION
►After primer removal is completed the lagging strand still contains gaps or nicks
between the adjacent Okazaki fragments. The enzyme ligase identifies and seals these
nicks by creating a phosphodiester bond between the 5' phosphate and 3' hydroxyl
groups of adjacent fragments.
► This replication machinery halts at specific termination sites which comprise a
unique nucleotide sequence. This sequence is identified by specialized proteins called
tus which bind onto these sites, thus physically blocking the path of helicase. When
helicase encounters the thus protein it falls off along with the nearby single-strand
binding
proteins.
DIAGRAMMATIC SEQUENCES OF DNA REPLICATION
Initiation
Primer synthesis
Synthesis of leading strand
Synthesis of lagging strand
Termination
TRANSCRIPTION
Transcription is the preliminary step of the gene expression that deals with the details
of the synthesis on mRNA (messenger RNA) from the promoter gene on the DNA
molecule under the agency of some enzymes & transcription factors. The enzymes
involved in transcription are called RNA polymerases. Prokaryotes have one type;
eukaryotes have three types of nuclear RNA polymerases.
RNA Synthesis
There are a number of different types of RNA, which play different roles in the cell:
♦ mRNA - encodes proteins.
♦ rRNA - forms the ribosome, including the active site for peptide bond formation.
♦ tRNA - adaptor, binds amino acids and rRNA and translates between mRNA and
protein.
♦ snRNA - small nuclear RNA, forms snRNPs, which process mRNA by removing
introns.
♦ snoRNA - small nucleolar RNA, forms snoRNPs, which process rRNA, mostly by
methylation and isomerisation.
♦ si RNA - small interfering RNA, involved in gene silencing and regulation.
♦ gRNA - guide RNA, needed for RNA editing, the removal and insertion of bases into
mRNA
♦ hnRNA - rag-bag of unprocessed pre-mRNA transcripts and other heterogeneous
nuclear RN As of less well defined function.
RNA polymerase
► RNA polymerase is the enzyme that generates RNA from DNA. Cells contain 20
times more RNA than DNA: in fact, about 5% of the cell is RNA, although only 5% of
this 5% is mRNA, because most of the RNA in the cell is rRNA.
► Since the majority of RNA is rRNA, Significantly more RNA is transcribed than
translated. This is especially true in eukaryotes, whose mRNA requires processing to
remove introns.
► The primary gene products of RNA polymerase (in eukaryotes) are:
• (pre-) mRNA (messenger);
• rRNA (ribosomal);
• tRNA (transfer);
• snRNA (small nuclear - spliceosomes);
• Other hnRNA (heterogeneous nuclear, such as snoRNA – small nucleolar).
Eukaryotic RNA Polymerases
Eukaryotes have three RNA polymerases which synthesized different type of RNA
 RNA polymerase I - rRNA.
 RNA polymerase II - mRNA.
 RNA polymerase III - tRNA
Steps Involve in Transcription of DNA to mRNA The overall transcription process required three distinct steps to complete Initiation
 Elongation
 Termination
Initiation
 Transcription actually starts from a very special region of DNA double helix
called “Promoter Region”, a region which has meaningful nucleobase sequences
of gene product (Protein).
 RNA polymerase with transcription factor (sigma factor) associates to the
promoter region to initiate transcription
Promoters
1. Only one strand of the DNA that encodes a promoter, a regulatory sequence, or a
gene needs to be written.
2. The strand that is written is the one that is identical to the RNA transcript, thus the
antisense strand of the DNA is always selected for presentation.
3. The first base on the DNA where transcription actually starts is labeled +1.
4. Sequences that precede, are upstream of the first base of the transcript, are labeled
with negative numbers. Sequences that follow the first base of the transcript, are
downstream, are labeled with positive numbers.
Steps of Transcription Process
Elongation
 RNA polymerase starst to move forward in a 5' to 3' direction. The polymerase
induces the 3' hydroxyl group of the nucleotide at the 3' end of the growing RNA
chain
which
attacks
(nucleophilic)
a
phosphorous
of
the
incoming
ribonucleotide.
 The complementary sequence of DNA come out as pre-mRNA during movement
of RNA polymerase
Termination
 The mechanisms by which eukaryotes terminate transcription are poorly
understood. Most eukaryotic genes are transcribed for upto several thousand
base pairs beyond the actual end of the gene. The excess RNA is then cleaved
from the transcript when the RNA is processed into its mature form.
 Finally the sigma factor & RNA polymerase remains separate as intact form.
Processing of RNA/ RNA splicing
The newly synthesized pre-mRNA contains “exon” & “intron” segment within it,
where the intron part has no impact on protein synthesis hence, a further processing
need to remove intron & make it as pure & mature m RNA. The process of cutting &
removing introns from pre-mRNA & joining the exons is referred as “RNA splicing”
RNA processing
Mechanism of RNA processing / splicing
The mechanism of RNA splicing is very complicated. The biochemical mechanism of
splicing consists of two reactionsFirst Reaction
In order to start the first reaction, the ending nucleotide of the intron react to first
nucleotide to form intron lariat, which will be remove at second reaction.
Second Reaction
Formed intron lariat then cut & removed from the pre-mRNA & exons are joined
together to form the mature or spliced RNA.
Mechanism of RNA splicing
TRANSLATION
Translation is a very key portion of central dogma, that deals with the synthesis of gene
product (protein) form spliced mRNA by using tRNA, ribosomal subunit & some
factors.
Steps Involve in Translation of mRNA to Protein
The overall mechanism of protein synthesis in eukaryotes is basically the same as in
prokaryotes, with three phases defined as initiation, elongation and termination.
However, there are some significant differences, particularly during initiation.
Translation of mRNA into a protein requires ribosomes, mRNA, tRNA, exogenous
protein factors and energy in the form of ATP and GTP.
Initiation
 Four major steps are required to initiate translation: ribosome dissociation,
formation of a pre-initiation complex, formation of the 405 initiation complex
and formation of the 805 initiation complex.
 The first step is the formation of a pre-initiation complex consisting of the 40S
small ribosomal subunit, Met-tRNAi met, eIF-2 and GTP;
 The pre-initiation complex now binds to the 5’ end of the eukaryotic mRNA, a
step that requires eIF-4F (also called cap binding complex) and eIF-3.
 The complex now moves along the mRNA in a 5’ to 3’ direction until it locates
the AUG initiation codon.
 Once the complex is positioned over the initiation codon, the 60S large ribosomal
subunit binds to form an 80S initiation complex, a step that requires the
hydrolysis of GTP and leads to the release of several initiation factors.
Elongation
 The elongation stage of translation in eukaryotes requires three elongation
factors, eEF-1A, eEF-IB and eEF-2, which have similar functions to their
prokaryotic counterparts EF-Tu, EF-Ts and EF-G.
 Although most codons encode the same amino acids in both prokaryotes and
eukaryotes, the mRNAs synthesized within the organelles of some eukaryotes
use a variant of the genetic code.
 During elongation the protein is synthesized one amino acid at a time on the 80S
ribosome. This process occurs in three major steps: binding of charged tRNA,
peptide bond formation, translocation of the growing peptide chain.
Termination
 When a stop codon appears at the A site translation is terminated. There are no
tRNA's that recognize stop codons.
 Instead releasing factors, eRF, recognize the stop codon. The releasing factors
along with peptidyl transferases and GTP catalyze the hydrolysis of the bond
between the polypeptide chain and the tRNA.
 The protein and tRNA disassociate from the P site and the ribosome dissociates
into the 405 and 605 subunits releasing the mRNA.
Diagrammatic sequence of Protein synthesis
References
1)
David Hames., Niger Hooper., Biochemistry- 3rd edition.
2)
Jeremy M. Berg.,John L. Tymoczko., Lubert Stryer. Biochemistry-5th edition.
3)
Robert K. Murray., Daryl K. Granner., Peter A. Mayes., Victor W. Rodwell.,
Harper’s Illustrated Biochemistry- 22nd edition.
4)
H.P.Gajera., S.V.Patel., Fundamentals of Biochemistry-A Textbook- 1st edition (2008).
5)
www.google.com (Wikipedia)
THE END