Lecture 10: transcription, translation and replication Flashcards
Where does transcription take place?
In the nucleus
Where does translation take place?
In the ribosome
Transcription
> Synthesis of RNA from a DNA template
-RNA is complementary to
DNA template
Catalysed by RNA polymerase
Transcription reaction equation
> Reaction is:
(RNA)n + ribonucleoside triphosphate <—-> (RNA)n+1 + PPi
RNA polymerase
> Catalyses the initiation and elongation of RNA chains
5’ –> 3’ polymerase activity
Requires:
-DNA template (double or
single stranded)
-All four ribonucleoside
triphosphates (ATP, GTP,
CTP, and UTP)
-Divalent metal ion (Mg2+
or Mn2+)
Stages of transcription
> Three phases: initiation, elongation and termination
Initiation occurs at the promoter
RNA polymerase and the sigma factor bind at the promoter
Terminator codes for a sequence that forms a hairpin structure followed by a sting of uridines
Initiation of transcription
> Stage 1: RNA polymerase binds to promoter
Stage 2: 17bp of DNA is unwound and transcription starts in ‘bubble’
Elongation: Stage one
Duplex DNA is unwound at the forward end of the RNA polymerase and rewound at the rear end. The RNA/DNA hybrid rotates rotates during elongation
Elongation: stage two
The RNA polymerase moves along the DNA. The length of the RNA-DNA hybrid is determined by enzyme, hybrid is separated and RNA leaves the enzyme.
Termination of transcription
> DNA template has start and stop signals
Termination involves several processes:
-Transcription stops
-RNA-DNA hybrid
dissociates
-Melted region of DNA
rewinds
-RNA polymerase releases
DNA
Types of RNA
> All types of RNA transcribed from a DNA template in manner described
Main 3 types of RNA:
-Messenger RNA (mRNA)
-Transfer RNA (tRNA)
-Ribosomal RNA (rRNA)
Messenger RNA (mRNA)
> Approx 5% of cellular RNA
No specific secondary structure (can form hairpin loops that regulate its lifespan)
Sequence of bases containing the information for the sequence of amino acids in the protein to be synthesised
-mRNA is the template for
protein synthesis
Variable size and sequence
Transcribed from protein-coding genes
The TATA box promoter- the main promoter in Eukaryotic cells
> This sequence is normally located 25bp upstream of the transcription site
It is AT rich and binds a number of proteins including RNA polymerase II
The proteins bind in sequence and do so to stabilise other proteins
Ribosomal RNA (rRNA)
> Approx 80% of cellular RNA
Several forms e.g. in prokaryotes have 23s, 16s, and 5s RNA
-called s because of their
sedimentation behaviour
-one molecule of each rRNA
species is present in each
ribosome
Major component of ribosome (the structures on which proteins are synthesised)
Folded into complex, 3D shapes
Transfer RNA (tRNA)
> Approx 15% of cellular RNA
Carries and delivers amino acids in an activated form to the ribosomes for peptide-bond formation during protein synthesis
At least one tRNA for each of the 20 amino acids
tRNA:
-an amino acid attachment
site
-a template-recognition site
is called an anticodon
Clover-leaf structure held together by hydrogen bonds between bases
Translation
> Synthesis of proteins
-the sequence of bases In
mRNA specifies the
sequence of amino acids
in the protein product
Takes place on ribosomes:
-large protein- rRNA
complexes
-two subunits of unequal
size
-subunits in prokaryotes
and eukaryotes differ
Binding site on ribosome
> Three binding sites:
-P (peptidyl- tRNA)
-A (aminoacyl-tRNA)
-E (exit)
The genetic code
> An amino acid is coded for by a group of 3 bases called a codon
There is no overlap of codons
The code is read continuously from a fixed starting point
The code is degenerate
The code is unambiguous
The code has start and stop signals
The code is almost universal
Degenerate definition
one amino acid may be coded for by several codons
Unambiguous definition
a codon only codes for one amino acid
tRNA
> Delivers amino acids to the ribosome
Each tRNA delivers a single, specific amino acid
Anticodon of tRNA recognises and base pairs with complementary code of mRNA
Wobble pairing of tRNA with mRNA
> There are 61 amino acids coding codons but only 40 tRNA molecules
Some tRNA recognise more than one codon (same amino acid)
5’ end of tRNA can have non-stranded base pairing
-wobble pairing
Initiation of translation
> AUG is initiation sequence
-methionine in eukaryotes,
formylmethionine (fMet) in
prokaryotes
-In prokaryotes, GUG can
also be initiation sequence
Translation does not start immediately at the 5’ end of the mRNA
-start is nearly always 25
nucleotides away from 5’
end
Reading frame Is established after the initiator AUG has been located
Initiation sequences
> Initiation sequence helps recruit the ribosome to the mRNA to initiate protein synthesis by aligning it with the start codon
Initiation sequences in prokaryotes
Needs purine-rich sequence on the 5’ side of the initiator sequence called Shine-Dalgarno sequence
Initiation sequences in eukaryotes
needs Kozak sequence followed by AUG (AUG closest to 5’ end)
Termination of translation
> Stop codes (UAA, UAG, and UGA) designate chain termination
These codes are not read by tRNA, but are read by specific proteins called release factors
-binding of the release
factor to the ribosome
releases the newly
synthesised protein
Translation occurs using multiple ribosomes
> mRNA is translates In the 5’ to the 3’ direction
Proteins are synthesised from the amino to the carboxyl end
Many ribosomes can translate a single mRNA at the same time
Enzymes involved in DNA replication
> In 1958 Arthur Kornberg and colleagues 1st isolated DNA polymerase from E. coli
DNA polymerase –> promote formation of phosphodiester bonds joining units of DNA backbone
The nucleotide added is the one able to form a base pair with the next nucleotide of the template
(DNA)n + dNTP <—> (DNA)n+1 PPi
There are several DNA polymerases
Can remove mismatched nucleotides in DNA (repair)
DNA polymerase 1 (Pol 1)
> 5’ —>3’ polymerase activity: addition of deoxyribonucleotides to the 3’ end of a growing DNA chain
DNA synthesis requires:
-the four deoxynucleoside
5’ triphosphate (dATP,
dGTP, dCTP, TTP)
-Mg²+
-a DNA template (is
template-directed enzyme)
-a primer strand with a free
3’-OH must already be
bound to the template
strand
Proofreading function of DNA polymerase 1
> Error correction in newly-synthesises DNA is a property of some DNA polymerases (not all)
-when an incorrect base
pair is recognised, DNA
Polymerase reverses its
direction by one base pair
of DNA
3’ –> 5’ exonuclease activity: Hydrolysis of terminal phosphodiester bond at 3’ end of DNA chain
-following base excision,
the polymerase can re-
insert the correct base and
replication can continue
Primer removal by DNA polymerase 1
> During DNA repair
5’ –> 3’ exonuclease activity: Hydrolysis of terminal phosphodiester bond at 5’ end of DNA chain
DNA polymerase III
5’ –>3’ polymerase activity
3’ –>5’ exonuclease activity
DNA ligase
Forms a phosphodiester bond between 3’ -OH at end of one DNA chain and 5’ -PO4 at start of another DNA chain. Joins two DNA chains together
DNA primase
Synthesises the primer- a short stretch of RNA
Helicase and gyrase
Unwinds the DNA by breaking hydrogen bonds between strands
DNA replication- leading strand
> Helicase unwinds DNA
DNA polymerase makes new DNA in 5’ to 3’ direction on leading strand
DNA replication- lagging strand
> On lagging strand:
-DNA polymerase cannot
synthesise in 3’ to 5’
direction
-Synthesis is in short
segments called Okazaki
fragments
Topoisomerases
> During unwinding, the DNA twists build up
-Build-up of twist would
form a resistance that
would eventually halt the
progress of the replication
fork
DNA topoisomerases are enzymes that solve these physical problems in the coiling of DNA
-Topoisomerase I cuts a single backbone on the DNA, enabling the strands to swivel around each other to remove the build-up of twists
-Topoisomerase II cuts both backbones, enabling one double-stranded DNA to pass through another, thereby removing knots and entanglements that can form within and between DNA molecules
Model of replication
> Replication starts at specific regions called the origins of replication
-Proteins are recruited to
this site forming bubble
Helicase and gyrase start unwinding double stranded DNA
Action of DNA primase
> DNA primase synthesises RNA primer on both leading and lagging strands
Action of DNA polymerase III
> Helicase moves along parental DNA unwinding it
DNA polymerase III catalyses synthesis of new DNA in 5’ —>3’ direction using 3’ -OH groups on RNA primers
Leading strand is synthesised continuously by DNA polymerase III
Lagging strand, primase repeatedly synthesises RNA primers which are then extended by DNA polymerase III
Joining Okazaki Fragments
> Requires DNA polymerase I and ligase
DNA polymerase I:
-Uses its 5’ to 3’ exonuclease activity to remove the RNA primers
-5’ to 3’ polymerase activity to fill the gaps left by removal of the RNA
Finally, DNA ligase joins the fragments
Origins of replication
> 245 bp region
Binding site for the helicase (4 separated repeats of a specific sequence)
Tandem array of 13 bp
-Sequence rich in AT codes
Single sites in prokaryotes
Eukaryotes have multiple points of origin
-Size of genome
-Decreased rate of
replication
Humans
-30000 origins of replication
-50-300kb
-Each origin activated
independently of its
neighbouring origin
Difference in eukaryotes?
> Enzymes known as DNA Pol α, β, δ (plus others)
Non-enzyme proteins: PCNA and RPA
these load DNA polymerase onto template and help it stay on/off
Histones?
> Nucleosome association conservative and dispersive
Quickly associated with new DNA
-15 mins fully packaged
H3 + H4 added to DNA within 1000bp of fork
Then H2a + H2b (<10000bp), H1
Telomerase- the enzyme
> Function: Replication of linear chromosome ends
Composition: RNA and protein
Protein- reverse transcriptase activity- copies RNA sequence
Binds to single stranded region (lagging strand) of linear chromosome and extends the ends (prevents chromosome shortening)
Transcription summary
> The information encoded in DNA is transcribed into messenger RNA (mRNA) by the enzyme RNA polymerase
Transcription occurs in the cell nucleus, where the DNA sequence is used as a template to synthesise a complementary RNA strand
The resulting mRNA carries the genetic code from the nucleus into the cytoplasm
Translation summary
> The information carried by mRNA is translated into proteins in the cytoplasm
Ribosomes read the sequence of nucleotides in mRNA in groups of three, called codons
Each codon corresponds to a specific amino acid, and the sequential arrangement of amino acids from proteins
Replication summary
> Genetic information is duplicated during DNA replication
The double stranded DNA molecule unwinds, and each strand serves as a template for the synthesis of a complementary strand
This process ensures the faithful transmission of genetic material from one generation of cells to the next
