Flashcards in Eukaryotic Transcription Deck (41)
What molecules catalyse transcription?
Define a gene.
A region of a genome transcribed into an RNA giving rise to a selectable phenotype.
What is a genome?
A collection of information-encoding transcription units
How do we measure where RNA molecules arise from a genome and the quantity produced?
Describe the steps of RNA-seq.
1) Purified from a cell
3) Reverse transcribed into cDNA
4) Sequenced using NGS
5) Aligned back to reference genome
What are the two main RNA-seq data outputs?
1) Plot distribution of sequence read frequency across the genome
2) Calculate a sum of the overall RNA level for the whole transcription unit (units: RPKM, FPKM or TPM)
What can the distribution of RNA-seq reads tell us?
The levels of RNA arising from a gene, variations in splicing and the start of transcription.
Why can RNA-seq not accurately determine the TSS of a gene?
Eukaryotic RNA molecules are processed.
What is NET-seq?
Nascent Elongating Transcription-seq is a modification of RNA-seq which allows us to catch an RNA pol and the nascent RNA in the act of transcription using anti-RNA pol antibodies. The nascent RNAs are purified and subjected to RNA-seq to reveal the 5' end of sequences of transcripts which can be mapped back to the genome to determine the TSS.
What are the different types of transcript-producing RNA polymerases in eukaryotes and where do they act ?
RNA poll I, II and III transcribe within the nuclear genome; bacteriophage-like RNA pols occur in mitochondria and chloroplasts; E.Coli RNA pol also occurs in chloroplasts.
What type of RNA does RNA pol I produce?
45S rRNA (around 80% of cellular RNA)
What type of RNA does RNA pol III produce?
tRNA and 5S rRNA (Around 15% of cellular RNA)
What type of RNA does RNA pol II produce?
mRNA, snRNA, snoRNA, miRNA and more (around 5% of cellular RNA)
What are the four phases of transcription?
RNA polymerases binding, initiation, elongation and termination.
How do eukaryotic RNA pols negotiate the phases of transcription?
They recognise specific sequence features in DNA using distinct additional factors/enzyme activities.
What is special about the Rpb1 subunit within RNA pol II?
It has a carboxy-terminal domain (CTD) which contains repeats of the heptapeptide sequence YSPTSPS (serines, threonine and tyrosine) that become reversibly phosphorylated to control which helper factors bind to RNA pol II.
What else is required for eukaryotic RNA pol binding or initiation at promoters?
What is the General/Basal Transcription Factor (GTF) complex?
The initiation factor for RNA pol II.
What makes up the pre-initiation complex (PIC)?
RNA pol II and GTFs.
What are the sub-complexes of the GTFs?
TFIID, TFIIB, TFIIF, TFIIE and TFIIH.
Which sub-complex of the GTFs recognises and binds promoter with TATA-Binding Protein (TBP) and up to 14 TBP-Associated Factors (TAFIIs)?
What roles does the TFIIB sub-complex of the GTF have?
Forms a physical bridge between TBP and RNA pol II and helps position DNA precisely into the Pol II active site.
Which GTF sub-complex promotes CTD-dephosphoryaltion, brings RNA pol II to the promoter as Pol II:TFIIF complex and stabilises the melted region of DNA at the TSS after TFIIE/H action?
What is the role of the TFIIE and TFIIH sub-complex of the GTF?
To melt the base-pairs within the TSS region using energy from ATP hydrolysis to create the transcription bubble ready for initiation; CTD kinase activity phosphorylates the CTD as transcription initiates.
How is the Transcription Elongation Complex formed (TEC)?
CTD phosphorylation (at Ser5, Ser7 and Tyr1) disengages the GTFs and allows elongation factors to bind instead.
How does termination of transcription occur for RNA pol II?
At the end of the coding regions the CTD phosphorylation pattern on RNA pol II changes (Ser2 is phosphorylated by CDK9) to allow binding of termination factors. Termination signal sequences, termed Poly (A) sites, at the end of the coding regions activate the CTD-associated cleavage and polyadenylation complex which cleaves the transcript to release the mRNA. CTD-specific phosphates including FCP-1 and Ssu72 dephosphorylate RNA pol II recycling it to bind with TFIIF for another round of transcription.
What are the unique features of eukaryotic rRNA transcription that allow sufficient quantities of rRNA to be generated?
Eukaryotic rDNA is an operon, rRNA genes occur in huge tandem repeats and rDNA genes use a relatively simple core promoter consisting of three main sequence elements.
What is meant by an operon?
More than one ultimate gene product is encoded in one poly-cistronic transcript.
How does termination of transcription occur for RNA pol I?
Terminator elements at the end of the rDNA coding region (TTF1 in mammals, Reb1p in yeast) cause RNA pol I to pause. The transcript is cleaved by the Rnt1 endonuclease and the polymerase is dissociated by Paused Transcript Release Factor.