RNA transcription Flashcards
What is transcription?
The process of RNA synthesis from a DNA template
The first step of ‘The Central Dogma’
What are the 3 main types of RNA?
What are they used for?
1) mRNA (messenger RNA)
- Codes for proteins
2) tRNA (transfer RNA)
- Participates in TRANSLATION
3) rRNA (ribosomal RNA)
- Major constituent of ribosomes
What is ‘The Central Dogma’?
A 2-step process in which the information in genes flows to proteins:
1) Transcription
2) Translation
What are ‘housekeeping genes’?
Required for the maintenance of cellular function
They are expressed ALL the time in cells
Are expressed in ALL cells of the organism
How much of the total RNA are is mRNA?
3-5%
How many families of tRNA are there?
49 families
What is the structure of tRNA?
Carries an amino acid
Has a specific anticodon loop to the codon on mRNA to ensure the right amino acid is added
How many types of rRNA are there?
4
How is the structure of RNA different to DNA?
1) Ribose sugar (instead of deoxy-ribose)
2) Uracil instead of Thymine
3) Synthesised as a single strand (from one DNA strand)
4) Very unstable (breaks down quickly)
What is the difference between a ribose sugar and a deoxyribose sugar?
In ribose - there is a -OH at 2 prime position on the sugar
In deoxyribose - there is -H at 2 prime position on the sugar
Describe the secondary structure of RNA
Base pairs with itself (folds in on itself)
Formation of stem-loop
Normal base pairing (A=U, G=C)
BUT many modifications also allow alternative base paring (non-Watson-Crick pairing (G=U))
Describe the tertiary structure of RNA
What does this allow?
Folding of a molecule in three-dimensions
Allows to have more of a function in the cell
Can form complex shapes which can bind and recognise other molecules
What are RNA polymerases?
Enzymes which perform RNA synthesis (transcription)
What are the 3 types of RNA polymerases in EUKARYOTES?
1) RNA polymerase I
2) RNA polymerase II
3) RNA polymerase III
What does RNA pol I make?
What is this substance involved in?
Ribosomal RNA (the bulk of all RNA in the cells)
Involved in translation
What does RNA pol II make?
Protein-encoding genes
Is involved in TRANSCRIPTION
What does RNA pol III make?
tRNA, small nuclear RNA and 5S rRNA (small RNA)
Which RNA type is regulated and why?
RNA pol II (involved in making protein-encoding genes through TRANSCRIPTION)
What does RNA pol II do?
1) Binds to DNA
2) Separates the DNA double helix
3) Adds ribonucleotide triphosphate
4) Moves along DNA in the 3’ to 5’ direction on ONE DNA strand (the TEMPLATE strand)
Which direction does RNA pol II synthesise RNA in?
5’ to 3’ direction
Which direction does RNA pol II move along DNA in?
3’ to 5’ direction
Making a 5’ to 3’ strand
How is the process of transcription different to DNA replication? (5 ways)
1) Multiple RNA polymerases bind on the same gene
2) No primer needed
3) Only ONE strand of DNA is used as the template
4) Transcript doesn’t remain bound to the template - immediately released from the ribosome
5) High error rate
Where are genes located on DNA?
What does this mean for RNA II?
On EITHER strand of DNA NOT overlapping (distinct regions)
RNA pol II can move on either strand of DNA
- BUT if moving left–> right - uses bottom strand
- Moving right –> left - used top strand
Is the template strand the ‘sense’ strand or the ‘anti-sense’ strand?
Why?
The anti-sense strand
To get a SENSE strand of DNA
What is the DNA strand NOT used by RNA pol II identical to?
The RNA made
What are topoisomerases and why are they needed?
Enzymes which cut one of the DNA strands to allow the strands to spin around and be re-joined
- Needed because as RNA pol moves along the DNA - every 10 bases, there is an extra coil induced into the DNA
- This provides resistance for the RNA pol to continue moving
SO, allows the RNA pol to progress
Where does transcription of a gene start? How?
At the promoter - non-regulatory proteins bind to the start site and tells DNA pol where and when to bind
Where is the TATA box found and what binds to it?
What does this cause?
- Upstream of the promoter for RNA pol2
- TATA binding proteins bind to it - distorting the DNA and bending it open
- Allowing RNA pol2 to bind to the DNA
What do general transcription factors do?
Recognise sequences in and around the promoter - guides RNA pol 2 to the right place on the DNA
Do GENERAL transcription factors have a regulatory role?
No
What is required for transcription to start?
A formation of a protein complex, consisting of:
- RNA polymerase
- GTFs
- Mediator
- Chromatin remodelling complexes
- Histone acetylases
- Histone-modifying enzymes
What is an enhancer?
A binding site for an activator protein, upstream of the transcription start site
What is the difference between general and specific transcription factors?
General:
- Not regulatory
- Required for the proper binding of RNA –> DNA
- Initiate transcription
Specific:
- Regulatory
- Bind to specific regions of the DNA sequence
- Increase the transcription of certain genes
Where does RNA processing happen and what are 3 RNA processing methods?
INSIDE the nucleus:
1) Splicing
2) Capping
3) Polyadenylation
Where does translation of RNA take place?
In the cytoplasm
Where are the untranslated regions of mRNA?
5’ - just after the cap
3’ - just before the polyA tail
What is splicing?
What does this form?
Elimination of the non-coding regions in pre-mRNA (introns)
Forms a MATURE mRNA
What is splicing specific to?
Eukaryotic mRNA
What is ‘alternative-splicing’?
When does this occur?
mRNA can be spliced in more than one way
When there is flexibility in where the introns are or when on gene codes for multiple DIFFERENT FORMS of a protein
What is the sequence of an intron?
2 guanines at either end - flanking the splice sites at the 5’ and 3’ end
Branch site in the middle of the intron - specific Adenine
What is the process of splicing?
1) 2 prime -OH on specific Adenine in the intron sequence attacks the phosphodiester bond between the 2 G’s at the 5’ end
2) Cleavage of the backbone
3) Connection of the G to the A - forms a lariat of the intron sequence
4) -OH on the the 3’ free end at G attacks the phosphodiester bond of acceptor G, at the start of the exon exon site
5) Joins the 2 exons together and removes the lariat, which is degraded
What is a lariat?
A loop of DNA
What is mRNA splicing carried out by?
The spliceosome (a nuclear complex)
What is the spliceosome made of?
About 150 proteins and 5 RNAs:
The core is formed by snRNPs
What are sRNAs?
What do they form?
Small nuclear RNAs: U1, U2, U4, U5, U6
Bind with many proteins to form snRNP (small nuclear ribonuceloproteins) to from the core of the spliceosome
What are the functions of the spliceosome/snRNPs
1) Recognise the donor and acceptor sites (nucleotide that specifies where splicing to occur)
2) Bring the 2 sites together (by bending RNA around)
3) Catalyse RNA cleavage (splicing)
Why is capping the 5’ end of RNA necessary?
For stability - prevents degradation
For biding of the mRNA to ribosomes and the initiation of translation
When is the 5’ cap added to RNA?
Early on in synthesis (when about 20-40 nucleotides long)
Which RNA is the 5’ cap added to?
Eukaryotic RNA
What happens to the 5’ end during capping?
1) 5’ end recognised by enzymes which bring in a guaniosine triposphosphate
2) 5’ end of guanosine connected to the 5’ end of RNA
3) Guanonsine is methylated
What is the difference between guanine and guanosine?
Guanine is the base
Guanosine is the nucleoside (guanine + sugar)
What is the difference between a nucleoside and a nucleotide?
Nucleoside = base + sugar Nucleotide = nucleoside + phosphate
What is the function of polyadenylation?
- mRNA stability
- Helps mRNA nuclear export and TRANSLATION
What is the poly-A signal?
AATAAA at the end of the gene (On DNA!!!)
Transcribes to AAUAAA on mRNA
What is the process of polyadenylatin?
1) Poly-A sequence in DNA is transcribed into the mRNA sequence
2) Poly-A sequence leaves pol II
3) CstF and CPSF immediately leave the CTD and bind to the poly-A sequence
4) RNA is cleaved and CstF falls from the poly-A sequence
5) Additional proteins assemble and create PAP (poly-A polymerase)
6) PAP adds ~200 A nucleotides to the free 3’ end
7) As the Poly-A tail is synthesised, poly-A binding proteins bind to it and decide the final length of the tail
8) PAP and CPSF drop off
What is the structure and function of the CTD (Pol II C terminal domain)?
Little ‘tail’ which is a part of the pol II protein structure
Highly phosphorylates to mimic the phosphorylated nature of the DNA
Serves to bind CstF (cleavage stimulating factor) and CPSF (cleavage and polyadenylation specific factor) which are waiting for the Poly-A signal in the mRNA to emerge from pol II
These proteins can immediately bind to the Poly-A site
Does PAP require a template?
No
What does CstF stand for?
Cleavage stimulating factor
What does PAP stand for?
Poly-A polymerase
What does CPSF stand for?
Cleavage and polyadenylation specific factor
What recruits PAP?
CPSF
What is the difference between coding and non-coding genes?
Coding genes are made into proteins
Non-coding genes are not made into proteins (they halt at mRNA) - not translated
