Download Medical Genetics, Lecture 3

Central Dogma of Molecular Biology
Dr.Aida Fadhel Biawi , October 2013
Enzymes in DNA replication
Helicase unwinds
parental double helix
DNA polymerase III
binds nucleotides
to form new strands
Binding proteins
stabilize separate
strands
Primase adds
short primer
to template strand
DNA polymerase I
(Exonuclease) removes
RNA primer and inserts
the correct bases
Ligase joins Okazaki
fragments and seals
other nicks in sugarphosphate backbone
Proofreading and Repairing DNA
• DNA polymerases
proofread newly made
DNA, replacing any
incorrect nucleotides
1 A thymine dimer
distorts the DNA molecule.
2 A nuclease enzyme cuts
the damaged DNA strand
at two points and the
damaged section is
removed.
Nuclease
DNA
polymerase
3 Repair synthesis by
a DNA polymerase
fills in the missing
nucleotides.
DNA
ligase
4 DNA ligase seals the
Free end of the new DNA
To the old DNA, making the
strand complete.
Accuracy of DNA Replication
• The chromosome of E. coli bacteria contains
about 5 million bases pairs
– Capable of copying this DNA in less than an hour
• The 46 chromosomes of a human cell contain
about 6 BILLION base pairs of DNA!!
– Takes a cell a few hours to copy this DNA
– With amazing accuracy – an average of 1 per
billion nucleotides
Protein Synthesis
• The information content of DNA is in the form
of specific sequences of nucleotides along
the DNA strands
• The DNA inherited by an organism leads to
specific traits by dictating the synthesis of
proteins.
• The process by which DNA directs protein
synthesis, gene expression includes two
stages, called transcription and translation.
Transcription and Translation
• Cells are governed by a cellular chain of
command
– DNA RNA protein
• Transcription
– Is the synthesis of RNA under the direction
of DNA
– Produces messenger RNA (mRNA)
• Translation
– Is the actual synthesis of a polypeptide,
which occurs under the direction of mRNA
– Occurs on ribosomes
Transcription and Translation
• In a eukaryotic cell the nuclear envelope separates transcription
from translation
• Extensive RNA processing occurs in the nucleus
Nuclear
envelope
DNA
TRANSCRIPTION
Pre-mRNA
RNA PROCESSING
mRNA
Ribosome
TRANSLATION
Polypeptide
(b) Eukaryotic cell. The nucleus provides a separate
compartment for transcription. The original RNA
transcript, called pre-mRNA, is processed in various
ways before leaving the nucleus as mRNA.
Transcription
• RNA synthesis
– Is catalyzed by RNA polymerase,
which Separates the DNA
strands and hooks together the
RNA nucleotides.
– Follows the same base-pairing
rules as DNA, except that in
RNA, uracil substitutes for
thymine
RNA
• RNA is single stranded, not double stranded like DNA
• RNA is short, only 1 gene long, where DNA is very long and
contains many genes
• RNA uses the sugar ribose instead of deoxyribose in DNA
• RNA uses the base uracil (U) instead of thymine (T) in DNA.
Synthesis of an RNA Transcript
• The stages of
transcription
are
– Initiation
– Elongation
– Termination
Promoter
Transcription unit
5
3
3
5
Start point
RNA polymerase
DNA
Initiation. After RNA polymerase binds to
the promoter, the DNA strands unwind, and
the polymerase initiates RNA synthesis at the
start point on the template strand.
1
5
3
Unwound
DNA
3
5
Template strand of
DNA
transcript
2 Elongation. The polymerase moves downstream, unwinding the
DNA and elongating the RNA transcript 5  3 . In the wake of
transcription, the DNA strands re-form a double helix.
Rewound
RNA
RNA
5
3
3
5
3
5
RNA
transcript
3 Termination. Eventually, the RNA
transcript is released, and the
polymerase detaches from the DNA.
5
3
3
5
5
Completed RNA
transcript
3
Synthesis of an RNA Transcript - Initiation
• Promoters signal the initiation
of RNA synthesis
• Transcription factors help
eukaryotic RNA polymerase
recognize promoter sequences
1 Eukaryotic promoters
TRANSCRIPTION
DNA
RNA PROCESSING
Pre-mRNA
mRNA
TRANSLATION
Ribosome
Polypeptide
Promoter
5
3
3
5
T A T A A AA
AT AT T T T
TATA box
Start point
Template
DNA strand
Several transcription
factors
2
Transcription
factors
5
3
3
5
3 Additional transcription
factors
RNA polymerase II
5
3
Transcription factors
3
5
5
RNA transcript
Transcription initiation complex
• Sense strand, or coding strand, is the
segment of double-stranded DNA running
from 5' to 3' that is complementary to the
antisense strand of DNA, which runs from 3'
to 5'. The sense strand is the strand of DNA
that has the same sequence as the mRNA,
which takes the antisense strand as its
template during transcription, and eventually
undergoes translation into a protein.
Promoter, a regulatory region of DNA
usually located upstream of a gene,
providing a control point for regulated
gene transcription .
Synthesis of an RNA Transcript - Elongation
• RNA polymerase synthesizes a single strand of RNA against the
DNA template strand (anti-sense strand), adding nucleotides to
the 3’ end of the RNA chain
• As RNA polymerase moves along the DNA it continues to
untwist the double helix, exposing about 10 to 20 DNA bases at
a time for pairing with RNA nucleotides
Non-template
strand of DNA
Elongation
RNA nucleotides
RNA
polymerase
A
3
T
C
C
A
A
3 end
U
5
A
E
G
C
A
T
A
G
G
T
Direction of transcription
(“downstream”)
5
Newly made
RNA
T
Template
strand of DNA
Synthesis of an RNA Transcript - Termination
•
•
Specific sequences in the DNA signal termination
of transcription
When one of these is encountered by the
polymerase, the RNA transcript is released from
the DNA.
Transcription Overview
Post Transcriptional RNA Processing
•
Most eukaryotic mRNAs aren’t ready to be translated into protein directly after
being transcribed from DNA. mRNA requires processing.
•
Transcription and RNA processing occur in the nucleus. After this, the
messenger RNA moves to the cytoplasm for translation.
•
The cell adds a protective cap to one end, and a tail of A’s to the other end.
These both function to protect the RNA from enzymes that would degrade
•
Most of the genome consists of non-coding regions called introns
1- Non-coding regions may have specific chromosomal functions or have
regulatory purposes.
2- Introns also allow for alternative RNA splicing
•
Thus, an RNA copy of a gene is converted into messenger RNA by doing 2
things:
– Add protective bases to the ends
– Cut out the introns
RNA Processing - Splicing
• The original transcript from
the DNA is called pre-mRNA.
• It contains transcripts of both
introns and exons.
• The introns are removed by a
process called splicing to
produce messenger RNA
(mRNA)
Alteration of mRNA Ends
• Each end of a pre-mRNA molecule is modified in a
particular way
– The 5 end receives a modified nucleotide cap
– The 3 end gets a poly-A tail
A modified guanine nucleotide
added to the 5 end
TRANSCRIPTION
RNA PROCESSING
50 to 250 adenine nucleotides
added to the 3 end
DNA
Pre-mRNA
5
mRNA
Protein-coding segment
Polyadenylation signal
3
G P P P
AAUAAA
AAA…AAA
Ribosome
TRANSLATION
5 Cap
Polypeptide
5 UTR
Start codon Stop codon
3 UTR
Poly-A tail
1 - Capping - the 5’ end of the pre-mRNA is capped with a 7methyl guanosine nucleotide.
Capping is required to:
1- Protect the RNA transcript from degradation
2-Plays an important role in mRNA transport to the
cytoplasm
3- Initiation of protein synthesis.
CAP
5’
3
’
2- Polyadenylation - the 3’ end of the pre-mRNA is
cleaved and a stretch of adenosines are added to the
end of the molecule.
Use of alternative polyadenylation sites can result in :
1- Insertion or deletion of sequences that control the stability of the
mRNA.
2-The polyA tail is also involved in translation termination.
CAP
5’
3’
An
3- Pre-mRNA splicing - intron-exon junctions (also
referred to as splice sites) in the pre-mRNA, the
intronic sequences are excised and the exons are
ligated to generate the spliced mRNA.
5’
CAP
An
3’
The enzyme that catalyzes intron excision is a
~ 3 MDa complex
spliceosome
pre-mRNA
spliced mRNA
Assembly and structural dynamics of the spliceosome, one of
the most complex molecular machines in the cell
However, multiple introns may be spliced differently in different
circumstances, for example in different tissues.
Heart muscle
1
1
2
2
Uterine muscle
5
3
3
1
3
4
4
5
5
Thus one gene can encode more than one protein. The proteins are
similar but not identical and may have distinct properties. This is
important in complex organisms
mRNA
Splicing
mRNA
mRNA
Typical human gene structure: 90-95% intron / 5-10% exon
average Intron 3500bp / average exon 150
Thus expression of a gene with an intron requires an extra step
to remove the intron
exon 1
DNA
intron
GE
exon 2
NE
transcription
pre-mRNA
intron
RNA splicing
mRNA
translation
protein
The human genome sequencing project (Venter
et al., 2001) estimated the number of human
genes to be between 30,000-40,000, which is
much less than the previous estimates .
There are 130 thousands types of proteins.
Types of RNA
Genetic information copied from
DNA is transferred to 3 types of
RNA:
mRNA
Copy of information in DNA
constitutes ( 2%) , 100,000 KINDS.
rRNA
Most of the RNA in cells is
associated with structures
known as ribosomes, the
protein factories of the
cells.(80%), FEW KINDS
It is the site of translation
where genetic information
brought by mRNA is
translated into actual
proteins.
Types of rRNA :
In prokaryotic :( 23S,16S,5S)
In Eukaryotic: ( 28S,18S, 5.8S and 5S).
Note: S ( Svedberg) is a unit refers to the
molecular weight and shape of the
compound .
tRNA
Brings the amino acid to the
ribosome that mRNA coded for.
(15%), 100 KINDS.
It has 4S in molecular weight.
Structure of tRNA