Eukaryotic Transcription

Question 1

What molecules catalyse transcription?

Answer 1

RNA polymerases.

Question 2

Define a gene.

Answer 2

A region of a genome transcribed into an RNA giving rise to a selectable phenotype.

Question 3

What is a genome?

Answer 3

A collection of information-encoding transcription units

Question 4

How do we measure where RNA molecules arise from a genome and the quantity produced?

Answer 4

RNA-seq.

Question 5

Describe the steps of RNA-seq.

Answer 5

RNA is…

1) Purified from a cell
2) Fragmented
3) Reverse transcribed into cDNA
4) Sequenced using NGS
5) Aligned back to reference genome

Question 6

What are the two main RNA-seq data outputs?

Answer 6

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)

Question 7

What can the distribution of RNA-seq reads tell us?

Answer 7

The levels of RNA arising from a gene, variations in splicing and the start of transcription.

Question 8

Why can RNA-seq not accurately determine the TSS of a gene?

Answer 8

Eukaryotic RNA molecules are processed.

Question 9

What is NET-seq?

Answer 9

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.

Question 10

What are the different types of transcript-producing RNA polymerases in eukaryotes and where do they act ?

Answer 10

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.

Question 11

What type of RNA does RNA pol I produce?

Answer 11

45S rRNA (around 80% of cellular RNA)

Question 12

What type of RNA does RNA pol III produce?

Answer 12

tRNA and 5S rRNA (Around 15% of cellular RNA)

Question 13

What type of RNA does RNA pol II produce?

Answer 13

mRNA, snRNA, snoRNA, miRNA and more (around 5% of cellular RNA)

Question 14

What are the four phases of transcription?

Answer 14

RNA polymerases binding, initiation, elongation and termination.

Question 15

How do eukaryotic RNA pols negotiate the phases of transcription?

Answer 15

They recognise specific sequence features in DNA using distinct additional factors/enzyme activities.

Question 16

What is special about the Rpb1 subunit within RNA pol II?

Answer 16

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.

Question 17

What else is required for eukaryotic RNA pol binding or initiation at promoters?

Answer 17

Initiation factors.

Question 18

What is the General/Basal Transcription Factor (GTF) complex?

Answer 18

The initiation factor for RNA pol II.

Question 19

What makes up the pre-initiation complex (PIC)?

Answer 19

RNA pol II and GTFs.

Question 20

What are the sub-complexes of the GTFs?

Answer 20

TFIID, TFIIB, TFIIF, TFIIE and TFIIH.

Question 21

Which sub-complex of the GTFs recognises and binds promoter with TATA-Binding Protein (TBP) and up to 14 TBP-Associated Factors (TAFIIs)?

Answer 21

TFIID.

Question 22

What roles does the TFIIB sub-complex of the GTF have?

Answer 22

Forms a physical bridge between TBP and RNA pol II and helps position DNA precisely into the Pol II active site.

Question 23

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?

Answer 23

TFIIF.

Question 24

What is the role of the TFIIE and TFIIH sub-complex of the GTF?

Answer 24

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.

Question 25

How is the Transcription Elongation Complex formed (TEC)?

Answer 25

CTD phosphorylation (at Ser5, Ser7 and Tyr1) disengages the GTFs and allows elongation factors to bind instead.

Question 26

How does termination of transcription occur for RNA pol II?

Answer 26

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.

Question 27

What are the unique features of eukaryotic rRNA transcription that allow sufficient quantities of rRNA to be generated?

Answer 27

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.

Question 28

What is meant by an operon?

Answer 28

More than one ultimate gene product is encoded in one poly-cistronic transcript.

Question 29

How does termination of transcription occur for RNA pol I?

Answer 29

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.

Question 30

What is needed for the RNA pol III pre-initiation complex?

Answer 30

TFIIIC and TFIIIB accessory factors. TFIIIC helps the TFIIIB complex bind upstream of the TSS which in turn helps RNA pol III bind at the TSS stabilised by Nab2.

Question 31

Does RNA pol III require the formation of a TEC?

Answer 31

No, the RNA pol III transcripts are too short to require the formation of a TEC.

Question 32

What facilitates RNA pol III termination?

Answer 32

RNA pol III itself: it recognises runs of 6-9 T bases a the end of the coding strand DNA which pauses the enzyme and allows subunit 11 to cleave and release the RNA. The Nab2 protein directs the RNA into the tRNA processing system.

Question 33

What is the common factor in promoter binding for all three nuclear RNA polymerases?

Answer 33

TATA box binding protein: generally binds AT-rich motifs in all promoters, binds in the DNA minor groove and bends the helix by 80 degrees.

Question 34

Compare eukaryotic nuclear transcription elongation.

Answer 34

RNA pol I and II use the same factors coordinated by CTD phosphorylation, RNA pol III is self-sufficient and there is no CTD phosphorylation.

Question 35

What is meant by endosymbiosis?

Answer 35

Chloroplasts and mitochondria were originally bacteria that entered a symbiotic relationship with archaeal-like cells and eventually become permanent fixtures.

Question 36

What are the features of human mitochondrial DNA (mtDNA)?

Answer 36

It is a circular molecule encoding 37 genes required for energy production that is transcribed as three operons each encoding a mixture of mRNA, tRNA and rRNA sequences which are separated by RNA processing steps.

Question 37

What is Mitochondrial RNA pol (POLRMT)?

Answer 37

A single polypeptide related to bacteriophage enzymes (transcription of the mitochondrial genome has stolen its mechanism from a phage infection within the endosymbiont)

Question 38

What makes up human mtDNA PICs?

Answer 38

POLRMT and two accessory factors including TFAM which bends DNA and probably wraps the promoter into a DNA loop around the PIC.

Question 39

What is the role of TEFM in human mtDNA transcription?

Answer 39

TEFM is a single polypeptide elongation factor that promotes elongation.

Question 40

What is the role of alpha-helical DNA binding proteins MTERFs in mtDNA transcription?

Answer 40

MTERFs act as regulated roadblocks to POLRMT enabling control over which component of an operon are produced (termination of mtDNA transcription occurs both within and at the ends of transcription units). They have functions in DNA replication and looping too.

Question 41

What are the features of chloroplast transcription?

Answer 41

Transcription uses both a bacteriophage-type RNA pol and an RNA polymerase with similar subunits to the E.Coli enzyme. Chloroplast genomes have multiple operons.