Eukaryotic Transcription and Gene Regulation Flashcards
Role of RNA Polymerase I
- transcribes ribosomal RNA (5.8S, 18S and 28S) in the nucleus
- generates components of ribosomes
Role of RNA Polymerase II
- transcribes all protein-coding genes, plus snoRNA, miRNA, siRNA, IncRNA, and most snRNA genes
- transcription takes place in the nucleoplasm
Role of RNA Polymerase III
- transcribes 5S rRNA, some snRNA, tRNA and other small RNAs in the nucleoplasm
- translation of mRNA into protein
Structure of the largest subunit in RNA Polymerase II
has a carboxy-terminal domain (CTD) consisting of multiple repeats of a heptamer
How many subunits are in EU RNA Polymerases
12 subunits and are complexes of ~500 KD
Stages of transcription
- enzyme binds to promoter and melts DNA
- enzyme remains stationary during initiation
- enzyme moves along template during elongation
- enzyme dissociates at termination
Events at template recognition stage
- RNA polymerase binds to duplex DNA
- DNA is unwound at promoter
Events at initiation
- chains of 2-9 bases are synthesised and released
Events at elongation
- RNA Polymerase synthesises RNA
- unwound region moves with RNA polymerase
- RNA polymerase reaches end of gene
Events at termination
RNA Polymerase and RNA are released
Requirements for transcription
- chromatin must be opened before RNA polhymerase can bind the promoter
- basal transcription factors
- coactivators
What are basal transcription factors
transcription factors required by RNA Polymerase II to form the initiation complex at all RNA Polymerase II promoters
RNA recognition to promoter
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)
Describe core promoter sequence elements bound by sigma factors
- 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
Transcription initiation in prokaryotes
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
Rho-independent termination
- 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
Rho-dependent termination
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
Organisation of genes in E.Coli
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)
“What is true for E.Coli is also true for the elephant”
Jacques Monod, 1972
Differences between PRO and EU gene transcription
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
Difference in the structure of EU and PRO
- EU organisms contain a membrane-enclosed nucleus, a mitochondrion, and a nucleolus
- PRO organisms contain a nucleoid, cell wall, flagellum, and capsule (sometimes)
Core promoter
recognised by general (basal) TFs that recruit RNA polymerase
Promoter proximal and distal elements
regulatory sequences and binding site for transcriptional activators and repressors (sequence-specific TFs)
Enhancer vs promoter
- 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
(EU) General TFs at core promoter
- 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
Sequence specific transcription factors that affect the rate of transcription initiation
- 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
DNA-binding of TFs
- via a specific hydrogen bond between an AA and a base
- a typical protein-DNA interaction might involve ~20 similar contacts
DNA-binding and dimerisation domains in TFs
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
How do transcription factors determine differential gene expression
LIVER CELL NUCLEUS:
- albumin is transcribed at a high level
BRAIN CELL NUCLEUS:
- albumin is transcribed at a low level
Chromatin remodeling
- 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
Processing of RNA transcripts in EU
- 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
Describe the ‘split’ of genes in EU
the coding sequences are interrupted with non-coding sequences (introns) that are spliced out of the mature mRNA transcript
Describe RNA splicing
- removes intron sequences from newly transcribed pre-mRNAs
- leaves the protein coding sequences (exons) in the mature mRNA
Describe the process of RNA splicing including the spliceosome
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
Describe the process of alternative splicing (differential splicing)
- 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
Export of mature EU mRNA from the nucleus
- 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
