Transcript lecture notes-molecular biology-web

Basics of Molecular Biology
Central Dogma:
- DNA replication
- Transcription
- Translation
Metabolic regulation:
- Genetic level
- Metabolic pathway control
- Cell receptor
Central Dogma
Central Dogma: universal
- Genetic information is stored on the DNA
molecule.
- This information can be replicated directly to
form a second identical molecule.
- Segments information on the DNA molecule
can be transcribed to yield RNAs.
- Using RNAs, this information is translated into
proteins.
DNA Replication
Preserving and propagating the cellular
message
Cell Division Cycle (Mitosis)
Fission
DNA Replication
DNA Replication : Preserve and propagate
the cellular message
DNA Replication
- DNA helix unzips and forms two separate strands.
- Each strand will form a new double strands.
- The two resulting double strands are identical, and
each of them consists of one original and one
newly synthesized strand.
- This is called semiconservative replication.
- The base sequences of the new strand are
complementary to that of the parent strand.
DNA Replication
- DNA helix unzips and forms two separate strands.
- Replication begin at the origin of replication –
predetermined site.
- Initiator proteins bind to the origin of replication of DNA
and break hydrogen bonds of base pairs in
the local region of the origin.
- The two strand separate to form
Y-shaped structure called a replication fork.
- Movement of the fork must be facilitated
by the energy-dependent action of
unwinding enzymes (helicase).
DNA Replication
- DNA synthesis requires:
- DNA template
- Activated monomers: nucleoside triphosphates:
dATP, dGTP, dTTP, dCTP.
- an RNA primer is synthesis by primase.
- DNA polymerase (Pol)
Pol III: mediates the addition of nucleotides
to an RNA primer.
Pol I: hydrolyze an RNA primer and duplicates
single-stranded regions of DNA polymer.
DNA Replication
DNA Replication
-
DNA synthesis:
- DNA polymerase works only in the 5’- to -3’ direction of the new
strand: the next nucleotide is always added to the exposed 3’—OH
group of the chain.
- The formation of the 3’, 5’ phosphodiester bond to link the next
nucleutide results in the release of a pyrophosphate
proving energy for such a synthesis.
- Leading strand: one strand can be formed
continuously if it is synthesized in the same direction
as the replication fork is moving.
- Lagging strand: the other strand must be
synthesized discontinuously.
- DNA ligase joins the two short pieces of DNA
on the continuous strand.
3’
5’
3’
DNA chain
http://www.ncc.gmu.edu/dna/repanim.htm
Transcription: sending the message
The primary product of transcription: m-RNA, t-RNA and r-RNA.
- RNA polymerases consist of :
sigma factor: a protein locate the beginning for the message.
core enzyme: it contains the active sites.
Read 3’ to 5’ direction of DNA template
RNA is synthesized in the direction of 5’ to 3’.
- Both strands of DNA could be transcribed.
- The base-sequence of RNA is the precise complement of the
DNA template sequence.
DNA template
RNA product
A
U
T
A
G
C
C
G
Transcription
Transcription requires:
- RNA polymerases
- Growth of RNA polymers is energy requiring.
- Activated ribonucleotide triphosphate:
ATP, GTP, UTP, CTP.
Transcription Process
- Initiation:
The sigma factor recognizes a specific sequence of
nucleotides on a DNA strand – promoter region.
the strands unwound.
- Elongation:
Transcription starts with the core enzyme then the sigma
factor is released.
- Termination:
RNA polymerase encounter a stop signal or transcription
terminator (in some case rho protein is required for
termination).
- the RNA polymerase dissociate from the DNA template
- the RNA transcript is released.
http://www.ncc.gmu.edu/dna/mRNAanim.htm
Difference in Transcription Between
Procaryotes and Eucaryotes
Procaryotes:
- Related protein are encoded in a row without
interspacing terminators.
- Transcription from a single promoter may result in a
polygenic message containing many genes.
- Regulation from a single promoter provide a efficient
regulation of functional related protein.
- No physical separation of chromosome and ribosome :
m-RNA bind to ribosome and begin translation while the
transcription is still on going.
http://micro.magnet.fsu.edu/cells/procaryotes/images/procaryote.jpg
Difference in Transcription Between
Procaryotes and Eucaryotes
Eucaryotes:
The DNA can encode for a transcript with an intervening
sequence called intron in the middle of the transcription.
- intron cuts out mRNA at two specific sites
- after it degraded, the spliced RNA fragments could be
joined by a process called m-RNA splicing.
- The spliced message can then be translated into an
actual protein.
- Once mRNA is recovered from the cytosol, it is mature
while it within the nucleus has introns.
- introns likely play a role in either evolution or cellular
regulation.
Difference in Transcription Between
Procaryotes and Eucaryotes
Eucaryotes: (continued)
Two modification of mRNA:
RNA capping: the 5’ end is modified by the addition of a
guanine nucleotide with a methyl group attached.
Polyadenylation: a string of adenine nucleotides are added
to the 3’ end. The string is several hundred nucleotides
long.
These two modifications are thought to increase mRNA
stability and facilitate transport across the nuclear
membrane.
Eucaryote cell structure
Translation
Translation is the final step on the way from DNA
to protein.
- It is the synthesis of proteins directed by a
mRNA template.
- The information contained in the nucleotide
sequence of the mRNA is read as three letter
words (triplets), called codons.
- Each word stands for one amino acid.
- During translation amino acids are linked
together to form a polypeptide chain which will
later be folded into a protein.
Translation: Message to Product
Universal three-letter codons on mRNA: A, G, C, U
- 64 codes for 20 standard amino acids
- more than one codon can specify a particular
amino acids.
- Nonsense codons: UAA, UAG and UGA
- Do not encode normally for amino acids.
- Act as stop points in translation.
- encoded at the end of each gene.
Translation
http://www.phschool.com/science/biology_place/biocoach/translation/init.html
Translation
Important Components:
Ribosome:
The ribosome is the cellular factory responsible for the protein
synthesis.
It consists of two different subunits, one small and one large
and is built up from rRNA and proteins.
sedimentation coefficients – rough mass determination:
30S (small subunits) and 50S (big subunits) for procaryotes
40S (small subunits) and 60S (big subunits) for eucaryotes
Inside the ribosome the amino acids are linked together into a
chain through multiple biochemical reactions.
Translation-components
t-RNA:
The charged t-RNA (aminoacyl-t-RNA) carries an amino
acid at one end and has a triplet of nucleotides, an
anticodon, at the other end.
It is formed by the energy from two phosphate bonds and
enzymes (aminoacyl-t-RNA synthetases)
The anticodon of a t-RNA molecule can basepair, i.e form
chemical bonds, with the m-RNA's three letter codon.
The t-RNA acts as the translator between m-RNA and
protein by bringing the specific amino acid coded for by
the m-RNA codon.
t-RNA
Translation-components
• m-RNA template
• The translation process also involves a
large number of protein factors that
facilitate binding of mRNA and tRNA to the
ribosome.
• Protein synthesis consumes a large part of
the energy produced in the cell.
Translation Processes
Translation consists of Initiation, Elongation and
Termination.
http://www.phschool.com/science/biology_place/biocoach/translation/init.html
Initiation results in the formation of an initiation complex in
which the ribosome is bound to the specific initiation (start)
site on the mRNA while the initiator tRNA charged with (Nformyl)methionine is annealed to the initiator codon and
bound to the ribosome.
- Protein synthesis begins with a AUG codon (less frequently
GUG) on the m-RNA
AUG encodes for a modified methionine, N-formylmethionine
(fMet).
In the middle of protein, AUG encodes for methionine.
Translation Processes
To recognize the initiation AUG:
Procaryotes: the AUG is preceded about ten nucleotides away
by a purine-rich RNA sequence that base-pairs with a
complementary sequence in a ribosomal RNA molecule.
5’
Purine-rich (-10)
AUG (+1)
fMet
m-RNA
Protein
Eucaryotes: the AUG closest to the 5’ end of an mRNA.
5’
cap
AUG (+1)
fMet
m-RNA
Protein
A procaryotic mRNA molecule can have a number of start sites
while a eucryotic mRNA has only one start site.
http://www.phschool.com/science/biology_place/biocoach/translation/init.html
http://www.phschool.com/science/biology_place/biocoach/translation/elong1.html
Translation Processes
Elongation joins amino acids to the growing
polypeptide chain according to the sequence
specified by the message.
- The formation of the peptide bond between the two
amino acids occurs on adjacent sites on the
ribosome: the P or peptidyl site and the A or
aminoacyl site.
- The growing protein occupies the P site, while the
next amino acid to be added occupies the A site.
- As the peptide bond is formed, the t-RNA
associated with the P site is released.
Translation Processes
Elongation (continued)
- As the peptide bond is formed, the t-RNA associated with
the P site is released.
- m-RNA was moved down one codon so as to cause the
t-RNA in the A site to be in the P site.
- The next charged t-RNA with the correct anticodon can be
recognized and inserted into the A site.
- The whole process is repeated until a nonsense code or
stop codon is reached.
- The cell requires four phosphate bonds to add one amino
acid to each growing polypeptide:
two to charge the t-RNA and two in the process of
elongation
http://www.phschool.com/science/biology_place/biocoach/translation/elong1.html
Translation Processes
Termination
- At a stop codon, a release factor reads the triplet,
and polypeptide synthesis ends.
- the polypeptide is released from the tRNA.
- the tRNA is released from the ribosome, the two
ribosomal subunits separate from the mRNA.
http://www.phschool.com/science/biology_place/biocoach/translation/term.html
Several ribosomes can translate an procaryotic
mRNA at the same time, forming what is called a
polysome.
More than one ribosome can translate an mRNA at one time,
making it possible to produce many polypeptides simultaneously
from a single mRNA.
Posttranslation
• Posttranslational modification means the chemical
modification of a protein after its translation. It is one of
the later steps in protein biosynthesis.
• It may involve the folding of a proper structure, the
formation of disulfide bridges and attachment of any of a
number of biochemical functional groups, such as
phosphate, acetate, various lipids and carbohydrates.
e.g. phosphorylation for controlling the behavior of a
protein, for instance, activating or inactivating an enzyme.
(TP53 function as tumor suppressor).
Posttranslation
• In procaryotes, posttranslation can take place
when proteins are secreted through a membrane
(cytoplasimc membrane or outer membrane)
(cotranslationally)
Such proteins exist in a pre-form from
translation which is the signal sequence plus the
mature form of protein.
A signal sequence is about 20-25 amino acids
and is clipped off during secretion.
Posttranslation
• In eucaryotes, proteins are released by exocytosis –
a process for a cell to get rid of the large molecules
through its membrane.
Transport vesicles
- carry proteins and other chemicals from
endoplasmic reticulum bound with ribosomes to golgi
apparatus and other membrane-bound compartment.
Posttranslation take places in these organelles.
- fuse with the plasma membrane
- release their contents
Posttranslation
- Only protein with a signal sequence can
enter secretory pathway.
- Two pathways:
- constitutive exocytosis: operates at all
time.
- regulated exocytosis: operates in
specialized secretory cells.
Posttranslation
• Some posttranslation can only occurs in
eucaryotes.
e.g.N-linked glycosylation involves in
endoplasmic reticulum and golgi apparatus.
It can serve to target the protein to a particular
compartment or to control its degradation and
removal from the organism.
Summary of Central Dogma
•
•
•
•
DNA replication
DNA-RNA transcription
RNA-Protein translation
Posttranslation
components, processes, features
Summary
DNA
RNA
Translation
replication transcription
Initiation
Elongation
(synthesis)
Termination
Components
(monomers and other
components)