The Mechanisms of Translation and Transcription Flashcards
What carriers out transcription?
DNA-dependent RNA polymerases (RNAPs)
Between which sites does transcription occur?
specific “start” and “stop” sites
At what part of the polynucleotide chain are nucleotides added?
the 3’ end of the polynucleotide chain
What synthesises RNA?
DNA-dependent RNA polymerases (RNAPs)
In which strand of the DNA is the genetic information transcribed into RNA?
the coding/sense strand
What does NTP stand for?
nucleotide triphosphate
How are NTPs selected for?
by base-pairing with the template strand
At which end of the extending RNA strand are NTPs added?
at the 3’ end
What does the RNA Polymerase active site contain?
a short RNA/DNA heteroduplex
What is transcription?
The process by which genetic sequence information that is stored in double-stranded DNA is copied into single-stranded RNA molecules
What do RNA polymerases do?
- join nucleotides together in a specific order
- determined by the sequence of the DNA
What is the process of transcription intrinsically dependent upon?
The DNA template
Where is transcription initiated?
specific transcription start sites
Where is transcription terminated?
specific transcription termination sites
In what direction is the RNA polynucleotide generated?
5’ to 3’
What must occur for complementary base-pairing to happen between DNA and RNA nucleotides?
a region of double-stranded DNA found within the active site of the DNA-dependent RNA polymerase is melted into constitutive single strands
How long is the transcription bubble?
approximately 10 nucleotides long
What does transcription produce?
an RNA transcript with the same genetic information sequence as the coding or sense strand of DNA
What type of chemical reaction occurs via the addition of a nucleotide to the RNA transcript?
a nucleophilic substitution reaction
What is the basic mechanism of this nucleophilic substitution reaction?
- 3’ OH group of the RNA attacks the alpha-phosphate group of the incoming NTP
- the oxygen atom connecting the alpha and the beta phosphate groups is displaced
- pyrophosphate (diphosphate ester) is released
How is Pyrophosphate/diphosphate ester abbreviated?
PPi
How does the 3’ OH group act as a nucleophile?
- has a lone pair of electrons
- giving it a partially negative charge
How does the alpha-phosphate of the NTP act as an electrophile?
- has a partially positive charge
- due to neighbouring electronegative oxygen atoms
In what direction are nucleotide sequences always written?
in the 5’ - > 3’ direction
What does the coding strand encode for?
encodes for the functional protein product of the gene
Are the template and coding strands gene-specific?
- yes
- it is only specific to that particular transcription “unit”
In what direction(s) can two RNA polymerases move along DNA?
- convergently (the RNA polymerases come closer together)
- divergently (RNA polymerases move away from each other)
Which sequence do we refer to when talking about the DNA sequence of a gene?
the coding strand’s genetic sequence
How does transcription termination occur in prokaryotic cells?
via destabilising the RNA/DNA heteroduplex
Which enzymes in prokaryotes can synthesise RNA in the absence of DNA?
- polynucleotide phosphorylase
- nucleotidyltransferase
- poly(A) polymerase
What is the E. coli RNA polymerase core enzyme?
- a protein complex containing 5 subunits
- a,a,ß,ß’,w
What do the beta subunits do in prokaryotic RNA polymerase?
- ß and ß’ have a catalytic function
- groove between the subunits allows for the binding of the double-stranded DNA
- where RNA synthesis takes place
What do the alpha subunits do in prokaryotic RNA polymerase?
- a and a bind transcription factors
- which regulates the enzymes activity
what does the omega subunit do in the prokaryotic RNA polymerase?
w is involved in the assembly and stability of the prokaryotic RNA polymerase
How are prokaryotic RNA polymerases targeted to transcription sites?
- via specific DNA-binding proteins
- sigma factors
What does the enzyme polynucleotide phosphorylase (PNPase) do?
- synthesise RNA
- requires nucleoside diphosphate (DNPs)
- functions equally well in the reverse reaction to degrade RNA
What do tRNA nucleotidyltranserases do?
- add a small number of specific nucleotides onto the 3’ end of tRNAs
- without a DNA template
- ensure tRNA molecules have the correct trinucleotide sequence at their 3’ end
What does poly(A) polymerase (PAP) do?
- extended 3’ ends of mRNA molecules with a stretch of A residues
- in the absence of a DNA template sequence
What is a general feature of gene expression in both prokaryotic and eukaryotic organisms?
the ability of particular proteins to recognise and specifically interact with defined nucleotide sequences within DNA or RNA
What does the protein sigma factor do?
- specifically recognises the sequence of NDA at the 5’ end of the gene
- so that RNA polymerase is correctly positioned to initiate transcription at the correct site
What is the region of DNA which RNA binds to to start transcription called?
the promoter region
How does the polymerase/sigma factor complex initially bind to the DNA?
- weakly and non-specifically
- so it can scan along the length of the DNA until it interacts with a gene promoter sequence
What happens when the RNA polymerase binds to the promoter gene?
- the RNA polymerase adopts an open and catalytically active conformation
What forms/happens once the RNA polymerase has switched to an open conformation?
- transcription bubble in the DNA binding site via the double-stranded DNA melting
- the enzyme synthesises a short RNA primer
When is the sigma factor released?
once the RNA/DNA heteroduplex is of sufficient length
What happens once the sigma factor is released?
- RNA polymerase moves away from the promoter
- RNA polymerase engages fully int he process of transcription
How is RNA polymerase different from DNA polymerase?
RNA polymerase has inherent DNA unwinding and primate activity
What is the stable interaction between the RNAP and DNA dependent on?
the DNA/RNA heteroduplex within the enzymes active site
What does destabilisation of the heteroduplex result in?
- the release of RNA polymerase
- transcription termination
What would continued transcription result in?
- block transcription events of other genes
- generate unwanted RNA sequences that would titrate out RNA binding proteins
- wasteful of important nucleotide resources
What is the least stable heteroduplex sequence?
A residues in DNA and U residues in RNA
What will a continuous stretch of A residues in the DNA template strand result in?
- a continuous stand of U residues in RNA
- this continuously unstable heteroduplex will destabilise the DNA/RNA interaction
- cause release of the RNA polymerase
What methods can be used in prokaryotes to terminate transcription?
- A-rich template strand DNA sequence
- Rho factor helices activity
What is Rho?
- a termination factor
- an RNA helices
How does Rho work?
- triggers the release of RNA polymerase
- uses the energy released through ATP hydrolysis to drive dissociation of the polymerase from the DNA
What are the 3 nuclear RNA polymerases present in eukaryotic cells?
- RNA polymerase I
- RNA polymerase II
- RNA polymerase III
What does RNA polymerase I synthesise?
- rRNA
- single transcript which is later process into 3 of the 4 rRNAs
What does RNA polymerase II synthesise?
- mRNA
- noncoding RNAs
What does RNA polymerase III synthesise?
- tRNA
- 5S rRNA
What do the 3 eukaryotic RNA polymerases have in common?
- common subunits
- a conserved structure which is homologous to prokaryotic RNA polymerase
What do the 3 eukaryotic RNA polymerases not have in common?
- unique subunits
- all 3 are visually different
- all 3 interact with distinct sets of genes
How are the genes within mitochondria and chloroplasts transcribed?
by additional RNA polymerases found within these organelles
How are eukaryotic RNA polymerases assembled at the promoter?
by protein-complexes / general transcription factors
How is eukaryotic RNA polymerase II assembly at the promoter initiated?
- transcription factor IID (TFIID) binds to an A/T rich sequence in the gene promoter region
- known as the TATA box
- through the TFIID subunit called TATA-box binding protein (TBP)
How does the TATA-box binding protein interact with the DNA?
- binds to the minor groove of the double-stranded DNA
What does the binding of the TATA-box binding protein to the DNA cause?
- the DNA to bend
- allowing the recruitment of further transcription factors
After more transcription factors are recruited to the TATA-box binding protein is bound what happens?
- preinitiation complex (PIC) is assembled at the promoter
- involving RNAPII and a number of general transcription factor complexes (gTFs)
Where does RNAPIII termination occur?
- at dA tracts in the coding strand
- resulting in transcripts having oligo (U) sequences at their 3’ end
How does termination of transcription by RNAPII occur?
- destabilisation of the polymerase/DNA complex
- through the action of exoribonucleases
the cleavage and polyadenylation complex endonucleolytically cuts the transcript and adds a poly(A) tail
What does the poly(A) tail of the RNA transcript allow for?
- binding of specific nuclear proteins which protect the released fragment from degradation
What happens to the 3’ fragment generated by cleavage?
- it contains an accessible 5’ end
- targeted by exoribonucleases which degrade downstream
- until the RNA polymerase is reached
- this displaces it from the DNA
In prokaryotes is translation and transcription couples?
- yes
- mRNA is translated into protein whilst it is being made by the RNAP
- allows for rapid gene expression
In eukaryotes is translation and transcription couples?
- no
- transcription occurs in the nucleus
- translation occurs in the cytoplasm
- allows for regulation at different steps in the gene expression pathway
