mol bio exam 2

5’ cap

• protects exonuclease and allows export from nuc

• capping proteins on ctd when ser2 unphosph

• cap as RNA comes out of machinery in initiation

3’ poly A tail

• helps RNA translocate

• splicing proteins move from CTD to RNA during elongation

• 3’ end processing proteins when Ser 5 unphosph

• add tail after cleavage

steps of 5’ capping

1. phosphatase removes phosphate

2. GTP adds GMP via reverse linkage

3. methyl transferase adds methyl

splicing

• introns are removed by the spliceosome + exons are joined tg to form mature mRNA

• requires a 5’ splice site, 3’ slice site, and branchpoint A that is far frrom donor

steps of splicing

A branchpoint attacks 3’ splice site and 3’ splice site attacks 5’ splice site

what is the spliceosome made of

snRNPs and snRNAs

snRNPs

complexes w RNA and proteins

snRNAs

define splice sites by base pairing w mRNA

constitutive splicing

splicing in normal fashion

alternative 5’SS

changes length of upstream exons

alternative 3’SS

changes length of downstream exons

what controls alternative splicing

1. proteins can bind to increase/ decrease splicing at specific sites

2. mRNAs can base pair with itself and prevent splicing at or near the location

3. histone modifications on nucleosomes can regulate splicing factors

4. methylated DNA can regulate splicing factors

RT-qPCR

PCR with RNA template —> measures amount of rna template

northern blot

1. separate RNA with gel

2. transfer to membrane and probe —> way to assess size

3. shows expression amount

tRNAs

• synthesized by RNA Pol III

• encoded by tRNA genes in DNA

• fold into 3D shape

• attached to aa by aaRS enzymes

• base pairs with mRNA in ribosome

tRNA processing

• 5’ leader removed

• intron removed by endonuc —> ends are ligated

• 3’ amino acid attachment site to help specific amino acids bind

aminoacetylation

• ATP —> AMP + 2Pi is used to create covalent bonds between aa and tRNA

• aaRS enzyme has active site that binds a subset of amino acids and editing sites removes wrong ones

• editing + active site —> 2 factor verification

composition of ribosomes

• core is made up of rRNA

• ribosomal proteins fill in crevices on surface

• RNA Pol I —> 29s, 18s, 5.85s

• Pol III —> 5s

• rRNA genes are repeated in genome

nucleolus

• ribosome producing factory

  • rRNAs

  • rRNA genes (rDNA)

  • RNA Pol I

  • snoRNPs

  • ribosomal proteins

  • small and large ribosomal subunits exist independently

role of snoRNAs

help w rRNA processing —> non-coding

initiation

mRNA exported to cytoplasm assumes a circular-like shape

1. small ribsomal subunit, eIF2, and GTP come together

   1. scan and find AUG

2. eIF2 hydrolyzes and initiation factors are released

3. large subunit binds

elongation

1. tRNAs are delivered to ribosome by EF-Tu (EF1)

2. correct match —> EF1 hydrolyzes GTP and unbinds —> aa-tRNA enters A site

3. peptidyl transferase forms peptide bond

4. EF2 (EF-G) binds to help move ribosome toward 5’

termination

1. release factor binds when stop codon is in the A site

2. water is added to the peptidyl tRNA instead of aa —> hydrolysis

3. ribosome disassembles and subunits bind diff mRNAs

reading frame

• out of frame —> wrong seq/ early stop codon

• ribosome cand change incorrectly added amino acids

• part of exons

global translation regulation

can be blocked by lack of eIF2-GTP

• needed for all translation

• if it’s phosph and left in GDP bound, GEF bound state —> unable to transcribe

• Gcn2 binds uncharged tRNAs and locks eIF2 (low [aa] limits translation process)

translation of an upstream ORF instead of protein coding ORF

• skip uORF if low [aa-tRNA]

• mRNA structure/ folding can change location of ribosome assembly

• proteins may bind to specific mRNA to block initiation at that location

ribosomes at premature stop codons —> stalling (why? + effect?)

• release factors work most efficiently near poly-A tail

• stalled ribosomes block upstream ribosomes from completing translation

stalled ribosomes —> mRNA degradation via nonsense mediated decay

anything upstream of exon junction complex signals premature stop codon

1. release factor binds at stop codon

2. if upstream of EJC, UPF proteins bind

3. translation terminates

4. UPFs recruit endonucleases, uncapping proteins, deadenylation proteins

5. mRNA is uncapped, deadenylated, and degraded by exonuc

ribosome profiling

1. RNAse cuts RNA thats not protected by ribosomes

2. isolate RNA + ribosomes they’re bound to

3. seq RNA + separate fragments via density

smORFs

• small open reading frames that encode f(n) proteins

• localized to organelles

miRNAs

• endogenous

• degrade mRNA and repress translation

• can interact w/ UTR and degrade it

• lin4 + lin-14 in bacteria while let-7 = conserved seq

biogenesis of miRNA

miRNA gene —> primary miRNA transcript via RNA Pol II —> drosha to precursor miRNA hairpin —> dicer cuts it —> joins miRISC and targets mRNA —> inhibits or degrades target mRNA w 3’ UTR

siRNA

• endogenous or exogenous

• responsible for mRNA cleavage + heterochromatin formation

• come from dsDNA

siRNA biogenesis

1. RNA transcription —> hairpin or 2 diff promoters

2. DICER —> 22 nucleotide dsDNA duplexes

3. loaded in siRISC with Ago

4. binds to target

5. Ago directly cuts mRNA in coding region

miRNA base pairing

imperfect BP allowed (ex: G-U allowed)

short seed seq —> initial anchor for miRNA-mRNA pairing

miRISC

1. removes poly A tail

2. recruits 3’-5’ exonucleases and degrades deadenylated mRNAs

3. recruits decapping complex and removes 5’ cap

4. recruits 5’-3’ exonucleases to recognize 5’ cap loss and degrade

how do 3’UTRs control gene expression

• have AAUAAA alternatives

• may cleave and polyadenylate early

• short 3’UTRs can’t be degraded with miRNAs

(proto)oncogene differences

• increased mRNA stability

• may favor translation of a real ORF

• increased protein stability

• alternative cleavage and poly-A sites are used more in cancer cells than non-cancer

3’ UTR implication in cancer

• short 3’ UTRs can’t be degraded with miRNAs

• miRNA genes can be considered oncogenes/ tumor supressors

• 3’ UTR can control protein-protein interactions —> functional complex