reaction mechanisms of replication, transcription, and translation (comparison of these)

all 3 have initiation → elongation → termination

Replication

DNA polymerase

more accurate repair

Transcription

• similar enzyme chemistry to DNA replication

Initiation

• cis elements in promoter are bound by transcription facots

• RNA polymerase is recruited

• TFIIB (e) or sigma factor (p) is displaced so that RNA can exit the polymerase active site via the RNA exit channel

Elongation

• Histones are moved and then replaced

• phosphorylated of CTD is necessary for this step

• polymerase “escape” from promoter allows this phase to happen

• transcription factors are replaced by elongation factors

• capping and splicing machinery are recruited to the CTD of the promoter and modify the RNA as it is being transcribed

Termination

• involves cleavage and polyadenylation of the emerging RNA molecule

• RNAse degrades the remaining RNA fragment, which is somehow ejected from the polymerase

Translation

• Four important components for translation

  • mRNA

  • tRNA

  • aminoacyl-tRNA synthetase

  • ribosome

• Three possible reading frames

  • the start codon determines the beginning of the aa chain, and thus the frame that will be used

  • 5’-AUG-3’ is almost always the start codon, but occasionally CUG, GUG, or UUG can be used instead

• polycistronic vs monocistronic

  • prokaryotes have many mRNA that are polycistronic

    • multiple ORFs

  • majority of eukaryotic mRNA are monocistronic

    • only one ORF

• prokaryotic mRNA

  • ribosome recruited by the Shine-Dalgarno sequence

    • also called ribosome binding site (RBS)

    • the sequence is complementary to one of the RNA components of the ribosome

  • the ribosome binds to RBS to orient it for translation at the start codon

• eukaryotic mRNA

  • ribosome recruited by the 5’ cap

  • ribosome scans for the “first AUG”

    • scans in the 5’ to 3’ direction on mRNA

    • surrounding nucleotides contribute to efficiency of translation

      • Kozak sequence is the consensus sequence surrounding and including the AUG (ATG in the sense strand of DNA)

• tRNA is an adaptor molecule

  • RNA nucleotide sequence needs to be converted to the appropriate aa sequence

  • tRNA is the bridge in between mRNA and nascent protein

    • specific tRNAs recognize the various codons in the mRNA sequence

    • a given tRNA is attached to a specific aa

• tRNA is L-shaped

  • the clover leaf diagrams represent well the base pairing within each of the stems/arms

  • x-ray structure of tRNA shows it is L-shaped

    • base pairing in stems

    • hydrogen bonding between each arm

• Unusual bases in tRNA

  • pseudouridine, dihydrouridine, thymine, methylguanine, and inosine

  • post-transcriptional modifications

  • tRNA bases are modified to stabilize the tRNA molecule’s structure, enhance its ability to recognize mRNA codons accuratley, and ensure efficient protein synthesis by facilitating propoer interactions with the ribosome, ultimately contributing to translational fidelity and accuracy

• common structure of all tRNAs

  • the acceptor stem

    • site of attachment to an amino acid

    • a common 3’ terminus

    • 5’-CCA-3’

  • ΨU loop

    • contains the pseudouridine (ΨU) modified base

  • D loop

    • contains the dihydrouridine (DHU) modified base

  • anticodon loop

    • responsible for base pairing with mRNA

  • variable loop

    • varies in size

• aminoacyl tRNA synthetase function

  • attaches amino acids to tRNA

  • charged tRNA = amino acid attached to tRNA

  • uncharged tRNA = no amino acid

• two steps performed by aminoacyl-tRNA synthetase

  • adenylylation

    • substrates

      • amino acids and ATP

    • products

      • adenylated amino acid and pyrophosphate

    • catalyst

      • aminoacyl-tRNA synthetase

  • tRNA charging

    • substrates

      • adenylated amino acid and uncharged tRNA

    • products

      • charged tRNA and AMP

    • catalyst

      • aminoacyl-tRNA synthetase

• 20 different aminoacyl-tRNA synthetases

  • there is a separate aminoacyl-tRNA synthetase for each amino acid

    • a given aminoacyl-tRNA synthetase can only add one type of amino acid to one or a few designated tRNAs

  • each aminoacyl-tRNA synthetase must recognize the following

    • their cognate amino acid

    • the appropriate tRNAs

• initiation

  • preparation of mRNA and the small subunit of the ribosome

    • initiator tRNA and many initiation factors (eiFs) assemble with the small subunit

    • many initiaion factors also assemble on mRNA

  • small subunit binds to mRNA

    • eIFs involved in recruiting the small subunit to the 5’ cap of the mRNA

  • scan for Kozak sequence with AUG

    • once found initiation factors will be rearranged

  • large ribosomal subunit binds to mRNA

• peptidyl transfer reaction

  • substrates

    • aminoacyl tRNA and peptidyl tRNA

  • peptidyl transferase reaction: nucleophilic attack of the amino group of aminoacyl tRNA

    • forms new peptide bound

    • breaks bond between peptidyl-tRNA and peptide chain

  • products

    • uncharged tRNA and a new peptidyl tRNA (with one additional aa)

  • catalyst

    • ribosome

  • three binding sites for tRNA in the ribosome

    • APE

      • A-site

        • binding site for the aminoacyl-tRNA

      • P-site

        • binding site for the peptidyl-tRNA

      • E-site

        • binding site of tRNA released from the polypeptide chain and about to exit the ribosome

    • these sites are located at the interface between the large and small subunit

  • entry and exit channels

    • mRNA entry and exit

      • located in the small subunit

      • entry channel only wide enough for unpaired nucleotides

    • peptide exit channel

      • located in the large subunit

• summary of termination in eukaryotes

  • stop codon enters A-site

  • release factor (eRF1) binds in A site

    • no tRNAs to bind the stop codon

    • eRF1 mimics tRNAs

  • release factor promotes hydrolysis of the peptide from the peptidyl tRNA

  • release factors and additional proteins contribute to ribosome disassembly/recycling

roles of general transcription factors for RNA pol II

• TFIID

  • TBP subunit (1)

    • recognizes TATA box

  • TAF subunits (11)

    • recognizes other DNA sequences near the transcription start point

    • regulates DNA-binding by TBP

• TFIIB (1)

  • recognizes BRE element in promoters

  • accurately positions RNA polymerase at the start site of transcription

• TFIIF (3)

  • stabilizes RNA polymerase interaction with TBP and TFIIB

  • helps attract TFIIE and TFIIH

• TFIIE (2)

  • attracts and regulates TFIIH

• TFIIH (9)

  • unwinds DNA at the transcription start point

  • phosphorylates Ser5 of the RNA polymerase CTD

  • relases RNA polymerase from the promoter

types of RNA made by RNA pol I, II, and III

RNA pol I: 5.8S, 18S, and 28S rRNA genes

RNA pol II: all protein coding genes, snoRNA genes, miRNA genes, siRNA genes, lncRNA genes, and most snRNA genes

RNA pol III: tRNA genes, 5S rRNA genes, some snRNA genes, genes for other small RNAs

role of CTD in RNA pol 11

• RNA pol that initially binds to the promoter is not phosphorylated

• the carboxy-terminal domain (CTD) of RNA pol gets phosphorylated in order for transcription to begin

  • TFIIH mediates the initial phosphorylation

  • leads to promoter escape by disrupting protein-protein interactions holding RNA polymerase at the promoter

• multiple phosphorylated residues are found in the tail of elongating RNA pol II

• polymerase phosphorylation state is also important in other stages of transcription and RNA processing

• CTD phosphorylation functions

  • in the transition from initiation to elongation, initiation factors are booted out and elongation factors are recruited

  • many aspects of the transition depend on the phosphorylation state of RNA polymerase

    • phosphorylation of RNA polymerase CTD (residue 5) by TFIIH facilitates promoter escape

    • P-TEFb is recruited, which phosphorylates an additional residue (residue 2) in the RNA polymerase CTD

    • elongation factors are recruited by binding to the phosphoserine residues

biochemical mechanisms of splicing

spliceosome

• splicing of mRNA via spliceosome is only known to occur in eukaryotes

• not all genes in eukaryotes are spliced

• 3 key sites important for splicing

  • 5’ splice site

  • branch site (branchpoint site)

  • 3’ splice site

• a polypyrimidine tract (C or U) is located in between the branch site and the 3’ splice site

• these sites direct where splicing will occur

• 2’ OH at branch site attacks phosphate in 5’ splice site

  • creates loop (lariat) in the intron

• 3’ OH of 5’ splice site attaches phosphate at 3’ splice site

  • this covanelntly and directly joins the two exons

• results in two products

  • spliced exons

  • intron lariat

• composed of RNA and protein components

  • more than 100 proteins

  • 5 RNAs

    • snRNAs

    • U1, U2, U4, U5, U6

• snRNA form RNA-protein complezes called small nuclear ribonuclear proteins (snRNPs)

• snRNPs recognize the splice sites through base-pairing of snRNA

  • the snRNA component of snRNPs can bind to the pre-mRNA splice sites

    • U1 and U6 can binds to the 5’ splice site

    • U2 binds to the branchpoint site

  • the snRNA can use base-pairing to bind snRNPs to each other

    • U6 and U2 bind to each other to bring together the 5’ splice site and the branchpoint

Splicing Mechanism

• U1 snRNP binds to the 5’ splice site, U2AF bind the 3’ splice site and polypyrimidine tract, and BBP binds the branch site

  • the early (E) complex

• U2 snRNP binds to branch site causing A (adenine) to bulge outward

  • the A complex

• tri-snRNP particle binds to the A complex

  • tri-snRNP = U4, U5, U6 snRNPs

    • U4 and U6 interact by base pairing

    • protein-protein interactions connect U5 to the complex

  • the B complex

• rearrangements within the B complex

  • U1 leaves the complex

    • U6 snRNP binds to the 5’ splice site

  • U4 leaves the complex

    • U6 snRNP binds to U2 snRNP

    • U6 snRNA also forms an internal stem loop

    • together U6 snRNP and U2 snRNP form the active site of the spliceosome

      • the 5’ splice site and the branch site are juxtaposed

  • the C complex

Splicing summary

• the U1 snRNP forms base pairs with the 5’ splice junction and the BBP (branch binding protein) and U2AF (U2 auxilliary factor) recognize the branch point site

• the U2 snRNP displaces BBP and U2AF and forms base pairs with the branch point site consensus sequence

• the U4/U6 U5 “triple” snRNP enters the reaction

  • U4 and U6 snRNAs are held firmly together by base pair intearctions

  • subsequent rearrangements break apart the U4/U6 base pairs, allowing U6 to displace U1 at the 59 splice junction

  • creates active site that catalyzes the first phosphoryl transferase reaction

self splicing introns - group II

• group II self-splicing occurs by a similar mechanism as the spliceosomal introns but do not require proteins

  • intron catalyzes reaction

• group II self-splicing introns are primarily found in bacteria and the mitochondria and chloroplasts of fungi, plants, algae, and some protists

• evolutionary significance

  • group II introns are though to be the evolutionary ancestors of the spliceosome

self splicing introns group I

• the mechanism of group I self splicing introns varies from. the canonical mechanism earlier

  • uses a gree G nucleotide (only hydrogen binding to RNA) to attack the 5’ splice site

  • the 3’ OH on the 5’ exon attacks the 3’ splice site

• Who has them?

  • fungi

    • found in the mitochondria of pathogenic fungi and are involved in regulating virulence and drug resistance

  • bacteria

  • eukaryotic microorganisms

    • many have them

    • tetrahymena have them in their ribosomal RNA

  • organelles

    • mitochondria and chloroplasts

  • bacteriophages

trans-splicing

• two exons from different pre-mRNA molecules are spliced together

  • rare

• uses the spliceosome

regulation of alternative splicing

mechanisms for mutually exclusive splicing

• steric hinderance

  • if the intron between the alternative exons is short, steric hinderance may prevent the binding of necessary splicing machinery

    • cannot fit the U1 snRNP at the 5’ splice site and the U2 snRNP at the branchpoint in the intron

• combinations of major and minor splice sites

  • splice site with sequences for the major spliceosome cannot be combined with sequences for the minor splieceosome

    • major (does most splicing)

    • minor

      • does less than 1% of splicing but defects in this system lead to disease

mutually exclusive exons + nonsense mediated decay = homeostasis

• nonsense-mediated decay

  • degradation of mRNA with a premature translation-termination (stop) codon

  • keeps the cell from having a bunch of truncated proteins that could have negative physiological consequences since all proteins bind other proteins

• dysregulation of this process is associated with a bunch of diseases as well as aging, but there is still much to be learned in this area

regulation by splicing activators and repressors

• the components of the splicoesome are expressed in all cell types

• various activators and repressors of splicing are expressed in specific types

  • allows for regulation of alternative splicing based on cell type

  • these regulators can interact with the spliceosome to affect its function

regulation of splicing

• cis elements involved in regulation

  • exonic splicing enhancers (ESE)

  • exonic splicing silencers (ESS)

  • intronic splicing enhances (ISE)

  • intronic splicing silencers (ISS)

• trans elements

  • activators

    • SR (serine-arginine rich) family

      • contains a domain for binding RNA (ESE or ISE)

      • domain for binding splicing machinery

      • function by recruiting splicing machinery

  • repressors

    • hnRNP (heterogeneious nuclear ribonucleoprotein) family

      • contains a domain for binding RNA (ESS or ISS)

      • functions by inhibiting the binding of splicing machinery

roles of 5' and 3' UTR in mRNA

• 5’ UTR binds CBP and eIFs

  • affects trafficking to cytosol and recruitment of ribosomes

• 3’ UTR affects half-life

  • poly-A tail and deadenulase

  • miRNA binding sites

translation initiation

• RNA polymerase binds to the promoter

• promoter determines where RNA polymerase will start transcription

• promoter contributes to regulating which cells will express that specific gene

• closed complex

  • when polymerase initially binds to the gene promoter

  • DNA is double-stranded

• open complex

  • DNA strands are separated around transcriptional start site

  • ~13 bp bubble

• initial transcribing complex

  • transcription of first 10 bp

  • transcription inefficient for these first 10 nucleotides

definitions of sense, antisense, upstream, and downstream

sense: template

antisense: matches copy

upstream: opposite of downstream

downstream: direction RNA polymerase is transcribing

• sense strand of DNA

  • same sequence as mRNA except T instead of U

• antisense strand of DNA

  • used as a template in transcription

• codon

  • the triplet in the mRNA

  • you can look these up in a codon table to determine the amino acid during translation

• anticodon

  • the portion of tRNA that base pairs with mRNA during translation