The genetic code and transcription III Flashcards
Expression of Genetic Information
Transcription process
Genetic info is encoded in DNA and this info directs the synthesis of proteins
> Information flow from DNA to proteins
> Transcription (RNA synthesis)
> RNA intermediate molecule
RNA intermediate molecule? makes sense. Why?
- DNA is chromosome bound (nucleus)
- But Protein synthesis occurs in ribosomes in cytoplasm
- RNA synthesis in nucleus and then….
- RNA migrates to cytoplasm
- Proportionally equal quantities of RNA and protein
All this suggests that genetic info on DNA is transferred to an RNA intermediate which then directs protein synthesis
RNA Polymerase
(Weiss, 1959)
Enzyme needed to prove that RNA can be synthesized from DNA template
= RNA polymerase (isolated from rat livers)
RNA polymerase has similar requirements to DNA polymerases;
1. DNA Template
2. All 4 nucleoside triphosphates - Ribonucleosides
(not deoxyribonucleosides)
3. Don’t need a primer for synthesis
(But polymerises in same direction as DNA - 5’→ 3’)
In Prokaryotes (E. coli):
- RNA polymerase has 5 subunits which forms the core enzyme
(aI, aII, b, b’, ω)
- A 6th subunit (Sigma (s)) is responsible for promotor recognition and binding. [regulates initiation, binding]
- When sigma binds to the core enzyme, then the RNA polymerase forms holoenzyme is formed
- Only one RNA poly. in bacteria, but some variability can result
from different sigma factors allowing the recognition of different
promoters
Eukaryotes have 3 types:
- RNA polymerases I, II, and III
- Each of which produce a specific type of RNA
- Each has more subunits than prokaryote RNAP
Transcription (RNA synthesis)
Components:
Transcription results in a single stranded RNA molecule that is complementary to one of the 2 DNA strands.
Strand from which the RNA is transcribed:
= Template strand
Other, non-template strand:
=Partner strand/ coding strand
Template binding:
Occurs when the sigma factor of the hollow enzyme recognises a specific DNA region
= The promotor
Promotors are known as cis elements
- Elements located on the same DNA molecule- elements made up of from the same DNA
Transacting factors:
- Molecules (like proteins) that bind to cis elements
- They recognise the cis elements and bind to the DNA
Promotors are located in the 5’ region
- upstream from transcription start site
- Promotors play an NB role in the regulation of gene expression since the interaction between the promotor and the RNA polymerase governs efficiency of transcription of that gene.
There are 2 NB consensus sequences present in the promotors of prokaryotic genes that have been strongly conserved during evolution
> -10 ( TATAAT, Pribnow box) - located 10 nucleotides upstream
from transcription start site.
> -35 (TTGACA, -35 region) - located 35 upstream
-Mutation in these 2 sites reduces transcription from the gene with a mutant promotor
- Degree of polymerised binding to different promotors varies
= results in variable gene expression
- These differences can be attributed to promotor sequence and mutations in promotors can drastically affect the initiation of gene transcription
- Strong and week promotors exist
> strong promotors can induce transcription as regularly as 1/2
seconds
> Weak promotors result in the initiation of transcription once,
every 20 min.
- Different sigma factors are also involved in the initiation of
transcription from different promotors - Most NB form of sigma factor = sigma 70 - Has a Mr of 70kDa
- Most prokaryotic promotors recognise sigma 70, however there are other sigma factors (other RNA polymerases) that recognise different promoter areas increasing the specificity of transcription initiation
Transcription process
- Template binding
RNA polymerase (sigma 70) recognises and binds to promoter - After binding, double helix is unwound to expose the template
strand –> using energy from ATP hydrolysis - RNA polymerase then initiates transcription by inserting the 1st 5’
ribonucleoside triphosphate – which is complementary to the
DNA at the start site (initiation; no primer) - More ribonucleotides are then added and linked by
phosphodiester bonds
= Chain elongation: 5’→3’ direction - This creates a temporary DNA-RNA duplex with anti-parallel
chains
- At this point, the sigma subunit is dissociated from the hollow
(core) enzyme - Chain elongation continues at a rate of (50 nucleotides/sec @
37°C) under the direction of the core enzyme. - At the end of the gene, the RNA polym. encounters a termination signal (hairpin loop or “r” / “rho” protein)
- The terminator contains a sequence which is repeated in
inverse a few base pairs later, and the inverse repeat is then
followed by a poly A region.
- When this is transcribed into mRNA, it causes the mRNA to form
a secondary structure, where the inverse repeats form hydrogen
bonds with their complimentary base pairs
- The loop structure that is formed, causes the RNA polym. to stall
over the poly A region of the DNA, and because the interaction
between A and U is weaker ( 2 H bonds) = Allows DNA-RNA
hybrid to dissociate
- RNA released and polymerase dissociates
When only this hairpin structure is needed for termination
=Intrinsic or rho independent termination
HOWEVER:
In some genes, termination needs a termination factor (rho)
- Rho factor binds to the end of the RNA chain and slides along
the strand towards the open complex bubble
- When the rho factor catches the RNA polym. (usually when it
stalls over the termination sequence) it causes termination of
transcription
- rho has an RNA helicase activity
> enables it to dissociate secondary RNA structures
(such as the hairpin loop) and also DNA-RNA hybrids
SO: by moving through the hairpin and dissociating the DNA template and the transcript - rho causes termination of transcription
polycistronic (prok) vs. monocistronic (euk) mRNA
In bacteria:
- Groups of genes whose products are involved in the same
biochemical pathway are often clustered together along the
chromosome = operons
- In most operons, the genes are contiguous
- Transcription begins at one initial transcription start site and
proceeds through the reading frames of multiple genes and its
only the final gene which contains the termination sequence
- As a result: transcription results in a large mRNA containing more
than one gene
=Prokaryotic mRNA = polycistronic mRNA
- It has multiple translation start sites for different proteins
transcribed by each gene
- Since the gene products or genes transcribed in this way are
usually needed at the same time - polycistronic mRNA provides a
proficient way to transcribe and translate the required info
- Mostly found in prokaryotes.
Eukaryotic mRNA = monocistronic
- only one single protein is encoded by each mRNA
- Has a single translation site
HAs been some exceptions in some of the lower eukaryotes.
Transcription in eukaryotes
Differences between prokaryotes:
1. Happens within the nucleus (3 RNA polymerases)
- before translation, mRNA must move into cytoplasm
2. Chromatin remodeling:
- Chromatin fiber must be uncoiled
- To give RNA polymerase and other regulatory proteins access to
the DNA
3. Initiation and regulation involves a more extensive interaction
between upstream DNA sequences and protein factors involved in initiation
(cis-acting DNA elements and trans-acting protein factors)
E.G: eukaryotic RNA polym. need the presence of several transcription factors to bind them in the correct place, relative to the transcription start site.
They may also need other enhancer and silencer factors
4. The primary RNA - produced by the RNA polymerase - must be modified to produce mature mRNA - which is eventually translated
- Primary mRNA transcripts are large – heterogeneous nuclear
RNA (hn-RNA). –> found only in the nucleus
- Of these only about 25% are converted to mature mRNA’s
5. Processing of primary transcript involves the addition of (5‘-cap and 3’-tail); and removal of part of the transcript (intron splicing) RNA editing
Promoters, Enhancers andTranscription factors - eukaryotes
There are 3 RNA polymerases in eukaryotes:
RNA polymerase I - forms rRNA - found in nucleolus
RNA polymerase II - mRNA, snRNA - nucleoplasm
- responsible for transcription of all eukaryotic mRNA.
- 2 large subunits and 10-15 smaller subunits
RNA polymerase III - 5s rRNA (smallest RNA), tRNA - nucleoplasm
RNPI and RNPIII involved in rRNA and tRNA transcription
Cis-acting elements in Euk. promotors
3 Cis-acting gene elements involved in euk. promotors
(core vs. enhancers):
- Core promotor - TATA box and CAAT box:
- Determines where RNA polymerase II binds to the DNA and
where it begins transcription - Enhancers (other regulatory elements) - they actually also
include suppressors as well (silencers)
- Regulate the efficiency/ rate of transcription initiation from the core promotor - The TATA box sits about 30 nucleotides upstream from transcription start site (-30)
- this sequence has a octanucleotide consensus sequence
= (TATA(A or a T)AAR) - R= any purine
- this sequence is analogous with prok –10; however:
RNA polym. binds directly to -10 seq. in prok, this isn’t the case for euk.- facilitates unwinding of helix; A=T
- 2 CAAT box:
- further upstream (-80);
- GGCCAATCT consensus
- Genes that have this element seem to need it for the gene to be transcribed in large quantities
- frequently absent in genes that encode for most proteins found in all cells. - Enhancers / Silencers:
- Affect the level of gene expression
Enhancers increase transcription, suppressors reduce transcription
modulate over a distance; position varies ( in comparison to core elements)
- can be placed anywhere
- Main function is to regulate transcription for that specific cells requirements. Or to ensure expression at a particular point of development or particular location
Trans-acting elements (eukaryotes)
- complement cis elements
- Facilitates RNA polym. binding and the initiation of
transcription - These proteins = transcription factors (TFs)
> General vs. specific TFs:
- General TFs:
- Needed for ALL RNA polym. II mediated transcription
Why are they so NB?
- Because in euk. - RNA polym. II cant bind directly to promotor
element
> Binding and initiation of transcription are dependent on
the general transcription factors to the core promotor
elements.
> they hold RNA polymerase II in the right place to begin
transcription
In humans: TFIIA en TFIIB
- TFIID binds directly to TATA box (≈sigma70)
- which is then followed by the sequential binding of the other
transcription factors and then RNA polym. II
= This forms pre-initiation complex - Specific TFs
= Transcriptional activators and repressors
- influence the rate/ efficiency of transcriptional initiation
- Bind to enhancer and silencer cis-elements
- either enhance or prevent pre-initiation complex - to increase/ decrease the rate of transcription
Processing of euk RNA (Caps & Tails)
In bacteria- the chromosome is located in the cytoplasm
- This makes entire process (transcription-translation) fairly simple and direct.
[Prokaryotes: straightforward –> DNA – mRNA – protein]
- many mRNAs can be transcribed simultaneously from the
same DNA molecule and ribosomes can attach to the still
growing RNA strands and can begin the translation process
while the mRNA is still being synthesised
In Eukaryotes: This CANT happen
- because the physical separation of the DNA from the
cytoplasm by the nuclear envelope
- the pre-mRNA also needs many stages of mRNA processing
before the mature mRNA is ready for translation
Process = post-transcriptional modification
Primary transcript versus mature transcript
- The primary mRNA - as directly transcribed by RNA polymerase II from the template strand, is considerably larger than the final mature mRNA - which is eventually translated into protein
mRNA Processing (5’Cap formation)
- Addition of a 7-methylguanosine (7mG) cap to the 5’ end of the
mRNA
- This can happen prior to the completion of the transcription of
the RNA molecule
- This helps protect the 5’-end against nuclease degradation -
since it is chemically similar to the 3’ end of the RNA
molecule
> This is because it is bonded in a special 5’-5’ way, to the
first nucleotide of the rest of the mRNA
= 5’ Carbon is bonded whilst the 3’ carbon is unbonded and maintains a hydroxyl end - which is similar to the 3’ ends of the rest of the mRNA
-Later the cap is involved in the transport of the mRNA, out of the nucleus, through the nuclear membrane into cytoplasm
- It is also involved in the initiation of protein translation
mRNA Processing (Polyadenylation)
- Addition of poly-A tail
- The 3’ end of mRNA’s contains up to 250 Adylinic acid
residues
= Poly-A tail - This sequence is added after the initial (primary) transcript is cleaved at a position 10-35 nt after AAUAAA
> AAUAAA = highly conserved polyadenylation signal - Mutations to the polyadenylation signal prevent further
processing of mRNA and the transcript is rapidly degraded - All eukaryotic mRNA (except histones) have such
polyadenylation signals and poly-A tails - Several viruses - especially plant viruses - also have
polyadenylated mRNAs - in order to better mimic their host
mRNAs
what is needed for the mRNA to be transported into the cytoplasm and translated
Both the 5’ cap and the 3’ poly-A tail are needed for the mRNA to be transported into the cytoplasm and translated
