lecture 1,2,3 Flashcards
why cells are so different?
although most cells in an organism contain the entire genome, only a small proportion will be expressed and this will govern the function of that cell
central dogma of molecular biology
DNA (gene) makes RNA makes PROTEIN, transcription then translation;
upstream 5’, downstream 3’
where RNA is made
nucleus; transcription
steps of eukaryotic transcription
- unwind dna and expose the basis on each strand (usually H bonded)
- one of dna strands acts as template
- transcription initiation complex
- addition of nucleotides on rna
- dna helix reforms behind
transcriptions initiation complex contains (3 parts)
rna pol 2 (enzyme, forms phosphodiester bonds and adds ribonucleotide) bound to a promoter (short dna sequence) and transcription factors (proteins)
rna pol 1, 2, 3 functions
size/weight
pol 1: synthesises rRNA
pol2: synthesises mRNA
pol3: synthesises tRNA, 5S rRNA, small nuclear RNAs
8-12 subunits, 500 kDa
template strand
3’ to 5’ that what rna polymerase uses
rna pol functions
unwinds and copies dna
more that 1 rna can be made from 1 dna
mRNA, rRNA, tRNA functions
mRNA (3%), code for proteins
rRNA(71%), forms the basic structure of the ribosome and catalyses protein synthesis
tRNA, adaptor between mRNA and aa (used in translation)
how does the cell know where on the dna to form the transcription initiation complex?
promoters and GTF
promoters, general characteristics and example
-short sequence found upstream of a coding region
-rna pol binds to dna
-like a little flag saying “im a gene, come and form initiation complex on me”
-TATA box (25-35 bp form the start site of transcription)
-people believed it was ridiculous that sequence was too short; it always starts with T (83%)
WHERE rna pol will bind
GTF
rna pol cannot directly bind to dna, GTF are needed
-transcribed by pol3, multimeric and highly conserved
HOW rna pol will bind
-gtf bind rna pol 2 at promoter site, resulting in TRANSCRIPTION INITIATION COMPLEX (TIC)
TIC
- GTF (proteins) + promoter (TATA box, short dna sequence) = TIC
assembly of TIC and start of transcription
- TBP (TATA-binding protein) binds to tata box in dna and bends the dna
- GTF: TF2D, TF2B, recruitment of rna pol 2+TF2F+CTD tail, TF2E, TF2H
- TF2H contains kinase and helicase activity-leads to phosphorylation of CTD
- rna pol 2 leaves the promoter and starts elongation of nascent dna
pre-mRNA/hpRNA
processing steps
gene copied with introns (non coding) and exons (coding) into rna steps: -5' capping -3' polyadenylation -splicing end: mature mRNA
advantages of polyadenylation
- stabilizing rna
- longer polyA tails=longer half life
- nuclear export
- translation
what is cDNA
copy of mRNA
elements for polyA (many A)
- polyA signal AAUAAA
- polyA site (rna will be cleaved)
- G/U region
factors for polyA
- CPSF with CStF, CF1 CF2 by bending pre-mRNA
- PAP (poly A polymerase), cleavage (split) 10-35 upstream of polyA signal causing slow polyadenylation (3’ polyadenylation); ENERGY DEPENDENT PROCESS (atp to ppi)
- after, CPSF CstF CF1 CF2 factors leave, and PAB2 binds. This increased the addition rate of A by PAP (rapid polyadenylation)
- 200-250 A: PAB2 signals PAP to stop
problems with gene expression lead to diseases
- cancer: absence of transcription off switch
- development disease: problems in expression of early proteins; like, kidneys when cannot be produced at all
- chronic disease: like, obesity; can cause inflammation and increase the risk of other diseases
where gene expression is regulated the most?
transcription initiation
two types of genes
constitutive (house-keeping): genes that most cells will need
highly regulated: selectively expressed under specific conditions (eg. cyclins-cell division) or in specific tissues
U1A
- nuclear protein
- involved in splicing of rna
- U1A binds its own mRNA, leading to inhibition of the polyadenylation meaning that pre-mRNA never matures
