Eukaryotic Transcription and Gene Regulation

Question 1

Role of RNA Polymerase I

Answer 1

• transcribes ribosomal RNA (5.8S, 18S and 28S) in the nucleus
• generates components of ribosomes

Question 2

Role of RNA Polymerase II

Answer 2

• transcribes all protein-coding genes, plus snoRNA, miRNA, siRNA, IncRNA, and most snRNA genes
• transcription takes place in the nucleoplasm

Question 3

Role of RNA Polymerase III

Answer 3

• transcribes 5S rRNA, some snRNA, tRNA and other small RNAs in the nucleoplasm
• translation of mRNA into protein

Question 4

Structure of the largest subunit in RNA Polymerase II

Answer 4

has a carboxy-terminal domain (CTD) consisting of multiple repeats of a heptamer

Question 5

How many subunits are in EU RNA Polymerases

Answer 5

12 subunits and are complexes of ~500 KD

Question 6

Stages of transcription

Answer 6

1. enzyme binds to promoter and melts DNA
2. enzyme remains stationary during initiation
3. enzyme moves along template during elongation
4. enzyme dissociates at termination

Question 7

Events at template recognition stage

Answer 7

• RNA polymerase binds to duplex DNA
• DNA is unwound at promoter

Question 8

Events at initiation

Answer 8

• chains of 2-9 bases are synthesised and released

Question 9

Events at elongation

Answer 9

• RNA Polymerase synthesises RNA
• unwound region moves with RNA polymerase
• RNA polymerase reaches end of gene

Question 10

Events at termination

Answer 10

RNA Polymerase and RNA are released

Question 11

Requirements for transcription

Answer 11

• chromatin must be opened before RNA polhymerase can bind the promoter
• basal transcription factors
• coactivators

Question 12

What are basal transcription factors

Answer 12

transcription factors required by RNA Polymerase II to form the initiation complex at all RNA Polymerase II promoters

Question 13

RNA recognition to promoter

Answer 13

1) RNA polymerase can bind DNA non-specifically, but cannot recognise the promoter
2) Holoenzyme can recognise and bind the promoter
3) Sigma factor binds consensus sequences in the core promoter (-35 and -10 sequence elements)

Question 14

Describe core promoter sequence elements bound by sigma factors

Answer 14

• consensus at -35 and -10 elements, which are the preferred places for sigma to bind
• there is spacing between these elements
• the more similar the consensus sequence is to the standard (e.g. TTGACA and TTATAAT), the better chance the promoter has of binding successfully and recruiting RNA pol

Question 15

Transcription initiation in prokaryotes

Answer 15

1) Open complex formation
- promoter opening allows for exposure of template strand for complementary RNA synthesis

2) Anchored transcription (abortive initiation)
- only short (abortive) transcripts can be synthesised, whilst Sigma factor remains bound to the promoter

3) Promoter ‘escape’ by RNA Polymerase
- Sigma factor released from promoter
- Elongation factors bind and transcription proceeds. The enzyme becomes processive and makes longer transcripts

Question 16

Rho-independent termination

Answer 16

• A terminating hairpin (palindromic sequence) forms on the nascent mRNA interacting with the NusA protein, causing RNA polymerase to stall
• The U-rich stretch downstream of the termination signal stimulates the release of the transcript from the RNA polymerase complex

Question 17

Rho-dependent termination

Answer 17

the Rho protein (RNA helicase) binds at the upstream rut site, translocates down the mRNA, and interacts with the RNA polymerase complex to stimulate release of the transcript

Question 18

Organisation of genes in E.Coli

Answer 18

FULL COMPLEMENTARITY
the coding sequences of genes are continuous (e.g. uninterrupted) and so transcript (RNA) is co-linear with gene (hybridisation with entire template DNA strand)

Question 19

“What is true for E.Coli is also true for the elephant”

Answer 19

Jacques Monod, 1972

Question 20

Differences between PRO and EU gene transcription

Answer 20

PRO:
- transcription/translation occurrence: at the same time in the cytoplasm
- gene structure: DNA sequence is read in the same order as the AA sequence
- no modification of mRNA after initial transcription before translation

EU:
- transcription/translation occurrence: transcription in the NUCLEUS and then translation in the CYTOPLASM
- gene structure: noncoding INTRONS with coding sequence
- modifications of mRNA: introns are spiced out; 5’ cap and 3’ poly A are added

Question 21

Difference in the structure of EU and PRO

Answer 21

• EU organisms contain a membrane-enclosed nucleus, a mitochondrion, and a nucleolus
• PRO organisms contain a nucleoid, cell wall, flagellum, and capsule (sometimes)

Question 22

Core promoter

Answer 22

recognised by general (basal) TFs that recruit RNA polymerase

Question 23

Promoter proximal and distal elements

Answer 23

regulatory sequences and binding site for transcriptional activators and repressors (sequence-specific TFs)

Question 24

Enhancer vs promoter

Answer 24

• enhancer contains several closely arranged sequence elements that bind TFs
• separationof enhancer from promoter may be several Kb
• promoter contains dispersed sequence elements that bind TFs; only the elements in the immediate vicinity of the startpoint for transcription are fixed in location

Question 25

(EU) General TFs at core promoter

Answer 25

- RNA Polymerase cannot find promoters independently - TFIID makes multiple specific contacts with core promoter elements, e.g. TBP (TATA box binding protein) interacting with TATA box; TFFID recruits TFIIB - TFIIB is critical for the recruitment of RNA Polymerase II (with TFIIF) - this complex cannot initiate transcription, even though RNA Polymerase II is there - once TFIIE and TFIIH have joined a transcription initiation competent pre-initiation complex is formed; TFIIE and TFIIH stimulate and stabilise the promoter opening, allowing for initiation of transcription

Question 26

Sequence specific transcription factors that affect the rate of transcription initiation

Answer 26

- sequences that bind specific TFs may be far from the actual transcription start site - DNA bending allows for specific transcription factors to interact with RNA polymerase complex and affect the rate of transcription

Question 27

DNA-binding of TFs

Answer 27

- via a specific hydrogen bond between an AA and a base - a typical protein-DNA interaction might involve ~20 similar contacts

Question 28

DNA-binding and dimerisation domains in TFs

Answer 28

a) helix-turn-helix motif b) zinc finger motif c) leucine zipper motif d) helix-loop-helix motif - dimers of the TF binds sequence-specifically to those short sequence elements, which are often arranged in a palindrome

Question 29

How do transcription factors determine differential gene expression

Answer 29

LIVER CELL NUCLEUS: - albumin is transcribed at a high level BRAIN CELL NUCLEUS: - albumin is transcribed at a low level

Question 30

Chromatin remodeling

Answer 30

- make promoter DNA accessible (prior to recruitment of RNA Pol) - chromatin remodeling complexes are recruited near gene promoters by TFs - remodelling of chromatin increases accessibility for transcription

Question 31

Processing of RNA transcripts in EU

Answer 31

- before translation - a 5' cap (modified GTP) is added at the 5' end; facilitates binding to ribosome and protects mRNA from being digested by ribonucleases - poly A tail (sequence AAUAAA) is added at 3' end after last codon; signals an enzyme to cut the pre-mRNA; signals enzyme to add 100-300 adenines (tail); the tail assists in export from nucleus and is important for the stability of the mRNA

Question 32

Describe the 'split' of genes in EU

Answer 32

the coding sequences are interrupted with non-coding sequences (introns) that are spliced out of the mature mRNA transcript

Question 33

Describe RNA splicing

Answer 33

- removes intron sequences from newly transcribed pre-mRNAs - leaves the protein coding sequences (exons) in the mature mRNA

Question 34

Describe the process of RNA splicing including the spliceosome

Answer 34

The spliceosome is a large RNA-protein complex that catalyses the removal of introns from nuclear pre-mRNA 1) snRNP molecules bind to consensus sequences in RNA near the 5' donor and 3' acceptor splice sites 2) binding of snRNA recruits other proteins 3) a cut is made between the 5' exon and the intron 4) after the first cut at the 5' end, the intron forms a closed loop 5) the free 3' OH group at the end of the cut exon reacts with the 5' phosphate of the other exon 6) the 3' exon is cleaved and spliced to the 5' exon and the mature mRNA is exported to the cytoplasm for translation 7) the excised intron is degraded in the nucleus

Question 35

Describe the process of alternative splicing (differential splicing)

Answer 35

- one gene, several proteins - alternative splicing can give more than one product from a single gene - alternative splicing can be regulated to give differential gene expression - e.g. striated smooth muscle, smooth muscle mRNA, fibroblast mRNA, brain mRNA

Question 36

Export of mature EU mRNA from the nucleus

Answer 36

- in PRO, RNA transcripts are translated while they are synthesised - in EU, RNA transcripts must be processed and transported to the cytoplasm before being translated. Mature mRNA leaves the nucleus through the nuclear pore complexes