Project Studying Synechococcus elongatus for biophotovoltaics

How to bioengineer a novel bio-photovoltaic system? Obtain a sequence by PCR, then clone it into a suitable plasmid We’re adding DNA, but want Synechococcus to make a protein!

Finding Orthologs Go to http://www.ncbi.nlm.nih.gov/ Enter name of gene in search window Select “nucleotide” Select name of a promising sequence Select “run BLAST”: optimize for somewhat similar sequences (blastn) Pick out interesting orthologs

Go to http://www.ncbi.nlm.nih.gov/ Select “structure” Finding Orthologs Go to http://www.ncbi.nlm.nih.gov/ Select “structure” Enter name of protein in search window Select name of a promising sequence Select “protein” Select “run BLAST” Pick out interesting orthologs Slr1962 protein TRAP dicarboxylate transporter

Cloning Orthologs Identify sequence from an organism that we can obtain For eubacteria DNA will do: no introns

Cloning Orthologs Identify sequence from an organism that we can obtain For eubacteria DNA will do For plants, animals, fungi or archaebacteria need mRNA

Cloning Orthologs Identify sequence from an organism that we can obtain For eubacteria DNA will do For plants, animals, fungi or archaebacteria need mRNA: DNA & 1˚ RNA contain introns

Regulating gene expression Goal is controlling Proteins How many? Where? How active? 8 levels (two not shown are mRNA localization & prot degradation)

mRNA PROCESSING Primary transcript is hnRNA undergoes 3 processing reactions before export to cytosol All three are coordinated with transcription & affect gene expression: enzymes piggy-back on POLII

mRNA PROCESSING Primary transcript is hnRNA undergoes 3 processing reactions before export to cytosol 1) Capping addition of 7-methyl G to 5’ end

mRNA PROCESSING Primary transcript is hnRNA undergoes 3 processing reactions before export to cytosol 1) Capping addition of 7-methyl G to 5’ end identifies it as mRNA: needed for export & translation

mRNA PROCESSING Primary transcript is hnRNA undergoes 3 processing reactions before export to cytosol 1) Capping addition of 7-methyl G to 5’ end identifies it as mRNA: needed for export & translation Catalyzed by CEC attached to POLII

mRNA PROCESSING 1) Capping 2) Splicing: removal of introns Evidence: electron microscopy sequence alignment

Splicing: the spliceosome cycle 1) U1 snRNP (RNA/protein complex) binds 5’ splice site

Splicing:The spliceosome cycle 1) U1 snRNP binds 5’ splice site 2) U2 snRNP binds “branchpoint” -> displaces A at branchpoint

Splicing:The spliceosome cycle 1) U1 snRNP binds 5’ splice site 2) U2 snRNP binds “branchpoint” -> displaces A at branchpoint 3) U4/U5/U6 complex binds intron displace U1 spliceosome has now assembled

Splicing: RNA is cut at 5’ splice site cut end is trans-esterified to branchpoint A

Splicing: 5) RNA is cut at 3’ splice site 6) 5’ end of exon 2 is ligated to 3’ end of exon 1 7) everything disassembles -> “lariat intron” is degraded

Splicing:The spliceosome cycle

Splicing: Some RNAs can self-splice! role of snRNPs is to increase rate! Why splice?

Splicing: Why splice? 1) Generate diversity exons often encode protein domains

Splicing: Why splice? 1) Generate diversity exons often encode protein domains Introns = larger target for insertions, recombination

Why splice? 1) Generate diversity >94% of human genes show alternate splicing

Why splice? 1) Generate diversity >94% of human genes show alternate splicing same gene encodes different protein in different tissues

Why splice? 1) Generate diversity >94% of human genes show alternate splicing same gene encodes different protein in different tissues Stressed plants use AS to make variant stress-response proteins

Why splice? 1) Generate diversity >94% of human genes show alternate splicing same gene encodes different protein in different tissues Stressed plants use AS to make variant Stress-response proteins Splice-regulator proteins control AS: regulated by cell-specific expression and phosphorylation

Splicing: Why splice? 1) Generate diversity 2) Modulate gene expression introns affect amount of mRNA produced

mRNA Processing: RNA editing Two types: C->U and A->I

mRNA Processing: RNA editing Two types: C->U and A->I Plant mito and cp use C -> U >300 different editing events have been detected in plant mitochondria: some create start & stop codons

mRNA Processing: RNA editing Two types: C->U and A->I Plant mito and cp use C -> U >300 different editing events have been detected in plant mitochondria: some create start & stop codons: way to prevent nucleus from stealing genes!

mRNA Processing: RNA editing Human intestines edit APOB mRNA C -> U to create a stop codon @ aa 2153 (APOB48) cf full-length APOB100 APOB48 lacks the CTD LDL receptor binding site

mRNA Processing: RNA editing Human intestines edit APOB mRNA C -> U to create a stop codon @ aa 2153 (APOB48) cf full-length APOB100 APOB48 lacks the CTD LDL receptor binding site Liver makes APOB100 -> correlates with heart disease

mRNA Processing: RNA editing Two types: C->U and A->I Adenosine de-aminases (ADA) are ubiquitously expressed in mammals act on dsRNA & convert A to I (read as G)

mRNA Processing: RNA editing Two types: C->U and A->I Adenosine de-aminases (ADA) are ubiquitously expressed in mammals act on dsRNA & convert A to I (read as G) misregulation of A-to-I RNA editing has been implicated in epilepsy, amyotrophic lateral sclerosis & depression

mRNA Processing: Polyadenylation Addition of 200- 250 As to end of mRNA Why bother? helps identify as mRNA required for translation way to measure age of mRNA ->mRNA s with < 200 As have short half-life

mRNA Processing: Polyadenylation Addition of 200- 250 As to end of mRNA Why bother? helps identify as mRNA required for translation way to measure age of mRNA ->mRNA s with < 200 As have short half-life >50% of human mRNAs have alternative polyA sites!

mRNA Processing: Polyadenylation >50% of human mRNAs have alternative polyA sites!

mRNA Processing: Polyadenylation >50% of human mRNAs have alternative polyA sites! result : different mRNA, can result in altered export, stability or different proteins

mRNA Processing: Polyadenylation >50% of human mRNAs have alternative polyA sites! result : different mRNA, can result in altered export, stability or different proteins some thalassemias are due to mis-poly A

mRNA Processing: Polyadenylation some thalassemias are due to mis-poly A Influenza shuts down nuclear genes by preventing poly-Adenylation (viral protein binds CPSF)

mRNA Processing: Polyadenylation 1) CPSF (Cleavage and Polyadenylation Specificity Factor) binds AAUAAA in hnRNA

mRNA Processing: Polyadenylation 1) CPSF binds AAUAAA in hnRNA 2) CStF (Cleavage Stimulatory Factor) binds G/U rich sequence 50 bases downstream CFI, CFII bind in between

Polyadenylation 1) CPSF binds AAUAAA in hnRNA 2) CStF binds; CFI, CFII bind in between 3) PAP (PolyA polymerase) binds & cleaves 10-35 b 3’ to AAUAAA

mRNA Processing: Polyadenylation 3) PAP (PolyA polymerase) binds & cleaves 10-35 b 3’ to AAUAAA 4) PAP adds As slowly, CFI, CFII and CPSF fall off

mRNA Processing: Polyadenylation 4) PAP adds As slowly, CFI, CFII and CPSF fall off PABII binds, add As rapidly until 250

Coordination of mRNA processing Splicing and polyadenylation factors bind CTD of RNA Pol II-> mechanism to coordinate the three processes Capping, Splicing and Polyadenylation all start before transcription is done!

Export from Nucleus Occurs through nuclear pores anything > 40 kDa needs exportin protein bound to 5’ cap

Export from Nucleus In cytoplasm nuclear proteins fall off, new proteins bind eIF4E/eIF-4F bind cap also new proteins bind polyA tail mRNA is ready to be translated!

Cloning Orthologs Identify sequence from an organism that we can obtain For eubacteria DNA will do For plants, animals, fungi or archaebacteria need mRNA 2) Identify coding sequence: Most sites identify Start and Stop codons We need 5’ and 3’ UTR!

Cloning Orthologs Identify sequence from an organism that we can obtain For eubacteria DNA will do For plants, animals, fungi or archaebacteria need mRNA 2) Identify coding sequence: Most sites identify Start and Stop codons We need 5’ and 3’ UTR! At Genbank or others can often obtain additional flanking sequence, eg, by clicking “CDS”

Cloning Orthologs Identify sequence from an organism that we can obtain For eubacteria DNA will do For plants, animals, fungi or archaebacteria need mRNA 2) Identify coding sequence: Most sites identify Start and Stop codons We need 5’ and 3’ UTR! At Genbank or others can often obtain additional flanking sequence, eg, by clicking “CDS” Or, get gene name and check at TAIR, MPSS or GRAMENE

Cloning Orthologs Identify sequence from an organism that we can obtain For eubacteria DNA will do For plants, animals, fungi or archaebacteria need mRNA 2) Identify coding sequence: Most sites identify Start and Stop codons We need 5’ and 3’ UTR! At Genbank or others can often obtain additional flanking sequence, eg, by clicking “CDS” Or, get gene name and check at TAIR, MPSS or GRAMENE If not, will need to obtain it based on position

Cloning Orthologs 2) Identify coding sequence: Most sites identify Start and Stop codons We need 5’ and 3’ UTR! At Genbank or others can often obtain additional flanking sequence, eg, by clicking “CDS” Or, get gene name and check at TAIR, MPSS or GRAMENE If not, will need to obtain it based on position 3) Design primers to obtain entire CDS + minimal flanking sequence from suitable source!

Cloning Orthologs Design primers to obtain entire CDS + minimal flanking sequence from suitable source! Get Primer 3 (or other program) to design primers that bind upstream of start and downstream of stop

Cloning Orthologs Design primers to obtain entire CDS + minimal flanking sequence from suitable source! Get Primer 3 (or other program) to design primers that bind upstream of start and downstream of stop: exclude the CDS http://primer3.wi.mit.edu/

Cloning Orthologs Design primers to obtain entire CDS + minimal flanking sequence from suitable source! Design nested primers that start at ATG and end at stop codon and add CACC at 5’ end

Cloning Orthologs Design primers to obtain entire CDS + minimal flanking sequence from suitable source! Design nested primers that start at ATG and end at stop codon and add CACC at 5’ end Probably will need to do this manually! Will break all the rules for primer design

Cloning Orthologs Design primers to obtain entire CDS + minimal flanking sequence from suitable source! Design nested primers that start at ATG and end at stop codon and add CACC at 5’ end Probably will need to do this manually! Will break all the rules for primer design May get away with it if only use them on amplicon

Cloning Orthologs Design primers to obtain entire CDS + minimal flanking sequence from suitable source! Design nested primers that start at ATG and end at stop codon and add CACC at 5’ end Probably will need to do this manually! Will break all the rules for primer design May get away with it if only use them on Amplicon Limited templates to bind

Cloning Orthologs Design primers to obtain entire CDS + minimal flanking sequence from suitable source! Design nested primers that start at ATG and end at stop codon and add CACC at 5’ end Probably will need to do this manually! Will break all the rules for primer design May get away with it if only use them on Amplicon Limited templates to bind 4) Do the two PCR rxns: if rxn 2 gives band of expected size clone it into pSyn_1/D-TOP