2.3 Transcription and translation Flashcards
What is transcription?
Copying of one strand of DNA into a complementary RNA sequence by the enzyme RNA polymerase.
What is RNA polymerase?
The enzyme that catalyses the synthesis of an RNA molecule on a DNA template from ribonucleoside triphosphate precursors
What are the types of RNA polymerases in both prokaryotes and eukaryotes?
What are the components of the prokaryotic RNA polymerase?
What is the holoenzyme?
When the core enzyme and sigma factor are together they are called the holoenzyme
Broadly what are the three stages of transcription?
- Initiation in prokaryotes
- Elongation
- Termination
Broadly what are the three stages of translation?
- Initation
- Elongation
- Termination
What is the process of transcription initiation in a prokaryote?
- The sigma factor binds to the promoter (2 highly conserved sequences)
- Recruites the core enzyme which melts the DNA, promoter is AT rich
- Start transcription
How does transcription initiation differ for eukaryotes and RNA polymerase II?
- The promoter is more varianle and complex than in prokaryotes
- The initiation complex involves many more proteins/factors
- General transcription facors
- RNA polymerase II
- Mediator complex
- Transcriptional activators
- Chromatin remodeling complexes
- Histone modifying enzymes
What is the purpose of general transcription factors in transcription initiation of eukaryotes?
They are needed to initiate transcription of all genes
TFII = Transcription Factors for Polymerase II
What does TFIID do?
I
What does TFIIH do?
- It has 9 subunits,
- It unwinds DNA at the transcription start point,
- Phosphorylates Ser5 of the RNA polymerase CTD;
- Releases RNA polymerase from the promoter
What is the process of transcription initiation in Eukaryotes?
- Transcription begins when TFIID binds to the TATA box via the TBP (TATA binding protein). THe TFIID complex also has 11 other proteins called TAFs (TBP-Associated Factor)
- Other GTFs and RNA polymerase II bind to form the pre-initiation complex
- Then TFIIH activates transcription by its helicase activity as it unwinds DNA at the TATA box and its kinase activity as it phosphorylates the CTD tail at Ser-5
- RNApol II now able to start transcription
What other factors are involved in the transcription initiation of eukaryotes?
What are some key components of the mediator?
What is the C Terminal Domain (CTD)?
- The CTD is a key regulatory region of RNA polymerase II which contains 53 tandem repeats of a 7 amino acid motif with Ser at position 2 and 5
- Phosphorylation of Ser5 affects the conformation and activity of RNA pol II releasing it to being transcription
- It contains proteins needed for RNA processing that will latch onto the tail
What is the process of elongation in transcription of eukaryotes?
- When Ser5 is phosphorylated, capping proteins bind to the CTD
- As the end of the nascent mRNA emerges the capping proteins can leave the tail and bind to the mRNA
- After about 20-30 nucleotides have been transcribed, capping enzymes add 7-methylguanosine to the end of the newly formed pre-mRNA 5’ end
- As elongation procedes, Ser2s starts to be phosphorylated by other kinases. This allows other proteins to bind such as splicing factors
- During elongation the phosphorylation of Ser5 is gradually lost and the proteins involved in the 3’ end processing bind to the CTD.
What is the process of termination of transcription for RNA pol II?
- Termination of transcription occurs when a specific sequence motif (AATAAA) occurs which is recognised by the Cleavage and Polyadenylation Specificity Factor (CPSF) and Cleavage Stimulation Factor (CstF) which ride on the CTD.
- The CPSF and CstF transfer to the mRNA and cause cleavage of the message
- Poly-A Polymerase (PAP) then adds multiple As to create the polyA tail. The Poly-A Binding Proteins (PABP) bind and protect it.
- More As are added to achieve a particualr length polyA tail for stability and funciton of the mRNA
How are introns defined by consensus sequences?
- Consensus sequences are recognised by proteins
- Must always have AG
What is the role of snRNP and the spliceosome?
- A snRNP is an example of ribonucleoprotein (RNA + protein).
- In snRNP the RNAs are small nuclear RNAs (snRNAs)
- A spliceosome is made of many snRNPs
What is the pre-mRNA splicing reaction?
- There is a particular A base in the intron that attacks the mRNA molecule at the splice junction and cuts it
- 3’ end of that molecule attacks the middle junciton and cuts it
- It gets ligated together and removed
What are some splicing errors?
- Some single nucleotide changes destroy a normal splice site causing exon skippings
- Some destroy normal splice sites and activate cryptic splice sites. mutate a splcie site so another not ideal one will be used
- Some create new splice sites that cause new exons to be incorporated
What is translation?
The process by which the sequence of nucleotides in an mRNA molecule directs the incorporation of amino acids into a protein polypeptide product. This requires amino acids, transfer RNA (tRNA) and is carried out by the ribosome
label tRNA
How is each amino acid covalently attached to its tRNA?
By a specific enzyme
What is a ribosome? and its subunits
- A ribonucleoprotein complex composed of rRNAs and ribosomal proteins that catalyses the synthesis of protein using information provided by the mRNA.
- There are two main subunits, the large (60S) and small (40S)
- Together they form the functional 80S ribosome
What are the three sites of a ribosome?
What is the process of translation initiation in eukaryotes?
What is the process of translation elongation in eukaryotes?
- Aminoacyl-tRNA binds and the first peptide bond forms
- The previous tRNA exits from the ribosome.
- The next aa-tRNA occupies the A site based on the 3 base codon sequence
- A new peptide bond is formed
- The 60S subunit moves forward followed by the 40S subunit
How is translation terminated in eukaryotes?
- The release factor binds to the A site when the STOP codon occurs.
- Finally the whole complex breaks up.
