Can the same enzyme bind to both 3' and 5'?
No
Bond formed by bases
H bond
Bond formed by sugar and phosphate
Covalent ( causes polarity)
DNA double helix features
antiparallel
complementary bases in each strand
1 helix turn =10bp -major/minor grooves
Draw sugar phosphate backbone
Configuration of nitrogenous bases
Planar
Where are the nitrogenous bases?
In the middle(creates favorable stacking energy bc planar)
Alpha helix vs DNA double helix
AA r groups stick out and bases point inward
Hydrogen Bond
Weak electrostatic interaction bc of polarity of covalently bound H and 2 EN atoms
What happens in hydrogen bonding?
H is cov bound to en atom and forms bond w another en H takes part + charge and en atoms part -
Strongest H bonds
Straight O—H|||||||O
H bond donors
OH or NH
H bond acceptors
O or N
Major/minor groove features
-glycosidic bonds don't directly oppose (asymmetry) -major groove can fit proteins
How do proteins find specific dsDNA sequences?
major groove -Can distinguish cg vs gc and at vs ta -determine bases through hbond donor/acceptor and methyl patterns
How do proteins access the bases to ID sequences?
DNA binding proteins ( read NA info) (TFs)
alpha helix of HTH domain fits in major groove
these proteins touch major and minor grooves
Multivalency
Build up of AA side chain interactions by DNA binding proteins (20+ weak contacts become strong)
Bonds of DNA binding proteins in the major groove
H bonding Ex: asp to A arg to G
Most common DNA form
B form
Other DNA forms
A form (wider and more compact) Z form ( left handed) 3strand(hoogsten bp)
RNA vs DNA
DNA: info storage Double stranded Antiparallel AT Deoxyribose Chargaff
RNA: info storage,info transport, catalysis Single or double or triple Parallel or anti AU No chargaff Ribose
What model describes the secondary structure of tRNA?
Cloverleaf
What base pairing does cloverleaf do?
Watson Crick ( can have open circles as long as base pairing maintained)
How is the tertiary structure of tRNA formed?
More H bonding (long range interactions) Non watson crick
How to clone a DNA fragment
Insert into vector and propagate in E. coli
Enzymes used to manipulate DNA
Nucleases DNA ligase DNA polymerase Polynucleotide kinase Phosphotase
What do nucleases do?
cut DNA
Endonuclease
Cleave dna internally by cutting phosphodiester bb
Exonuclease
Cuts DNA molecule on either end
DNA ligase
Glue Join DNA molecule end to end by catalzkng phosphodiester bond between 3' OH and 5' PO4
DNA polymerase
Synth DNA from template strands
Polynucleotide kinase
add triphosphate to 5' OH
Phosphatase
Removes 5' triphosphate
Restriction enzyme
Endonucleases in prokaryotes that fight over invading viruses recog and cleaving dna seqs
Modification enzyme
Modifies host dna through methylation (restriction enzymes with cut unmethylated dna)
How to restriction enzymes recognize their sites?
*Bind to DNA and scan for recog seq (bind will change the DNA conform) *multiple backbone contacts with major and minor grooves *AAs h bond to phosphates in bb and bases They cut at specific sequences which are usually 4, 6, or 8 base palindromes Frequency of restriction sites is 4^n (n=bases)
Popular digestion enzyme
HindIII
How do dna frags reanneal?
Bc of sticky ends ( overhanging bases) Ligase will glue the breaks with phosphodiester bond in bb
What are the necessary features of a vector?
origin of replication (sequence that allows replication in e coli cell) -selectable marker ( gene to tell which bacteria have the vector)
can accom additional DNA ( big and small frags)
unique restriction site where foreign DNA is inserted
How does gel electrophoresis separate DNA frags?
Fragments migrate according to size (constant mass charge ratio) Diff agarose concentrations allow diff band resolutions
Which gels for large and small frags?
Large: agarose 100nt-10kb Small: acrylamide 10-50nt
How to create a human DNA library?
isolate DNA from lymphocyte and part digest with HindIII and digest vector with HIII
mix vector + insert and DNA ligase
transform DNA into e coli and select for recomb DNA (on agar with selection compound and galctosidase substrate
Use double selection
Or just use PCR
cDNA library vs genomic library
Cdna: cloned rev transcribed mRNA ( no introns expressed genome ) - if genomes or related is known can map to referemce Genomic: large DNA frags ( whole genome) - want to do if genome seq is unknown
How to design primers to amp dna seq?
What must a DNA strand have to grow?
3' OH group (primers have this)
How are dideoxynucleotides used in dna seq?
Sanger sequencing Replication stops w/o 3' OH *We use them to block further DNA molecule growth Add to reaction mix has H instead of OH on 3' and prevent phosphodiester bonding
Draw dieoxynucleotide
DNA synth direction
5 to 3 Only add new subunits to 3'
How can you use short seq to build a complete seq?
Short overlapping seqs come together as contigs Software will overlap snd configure
How big is the human genome?
3x10^9 base pairs ( billion)
C-value paradox
lack of correlation between genome size and the biological complexity of an organism (we don't know why)
Hyperchromisity
Single stranded dna absorbs higher uv than double (cot)
Cot procedure
1 shear dna into fragments 2 denature at 95 c 3 reassociate through slow cooling at different concentrations (Co) and rates of time (t1/2) 4 measure percent reassociated to ds
Cot anaylsis
Simple dnas/more repeated sequences reassociate faster -larger genomes are slower Human DNA is different because different fragments reassociate as different times Unique seqs long time
ORF qualifications
Over 100 AAs Start atg
E. Coli genome features
Circular dna 1 origin if rep Wall to wall genes No introns Operons Few repeats Horizontal gene transfer recent
How do you find genes in a primary sequencing read?
Prok:easy bc no introns, count in between and stop at the right stop, look for recognizable promoter Euk: hard, scan with computer find start and try to count 100 triplets before stop codon
Yeast genome features
Wall-wall genes 16 chrom, mito, and multiple origins No operon Some repeated genes A few introns More gene density than humans
How many genes in the human genome?
20,000 (splice to make more proteins)
What percent of our genome is related to gene control and what percent encodes for proteins?
20 percent & 1-1.5
How many open reading frames in the human genome?
About 20,000
Simple repeats
Repeated seqs 1-30 nuc found between or inside genes
Types of simple repeats
Mononucleotide Dinucleotide: highly polymorphic ( good genetic markers ) Trinucleotide: disease causing VNTRs: good at iding individuals
Which simple repeat causes diease?
Trinucleotide REPEATS EXPAND WITH EACH GENERATION( need to relax dna repair fidelity) CCG or CAG in coding or non coding dna are linked to disease like huntingtons (more is higher risk)
DNA only transposons: stucture frequency movement
Cut and paste genes from one area of the genome to another until they become inactive (fossilized)
LINES: structure frequency movement
20% of genome Most abundant class of repeated DNA 1-9kb L1 most common 6.5 kb (only 50 functional rest only transpose) Non retroviral transpons L1s move when transcribed by rna poly II to make mrna which is translated into nuclease and reverse transcriptase copies it back to dna
Which repeated DNA encodes reverse transcriptase and endonuclease?
LINES
SINEs
13% of genome 10^6 Alu element is most common (restriction site for RE Alu1)
300 nuc, poly A tail, GC rich Use same reverse transcriptase as LINEs Transcribed by rna pol II but don't encode, just make small tRNA Alu element looks like 7s rna
Why can SINES cause diease?
.01% disease The alu elements can be insertional mutagens when they jump Our enzyme will methylate the C in the CpG gene which decreases transcription ( turns off genes )
Third most abundant class of repeated DNA
Retroviral like elements
Types of satellite DNA
Centromere and telomere # repeats can change from uneven crossing over
Repeated genes and how they arose
mRNA, tRNA, histones Duplicated during evolution bc cell needs many copies Globin, growth hormone, and albumin common ancestors 1 gene duplicates and both may slight change
Pseudogenes
former genes that have accumulated mutations and are nonfunctional
How do genes duplicate???
Unequal crossing over, slippage
Reverse transcriptase mechanism
Copies RNA into DNA Host uses no such enzyme so good inhibitor target (HIV) adds nuc onto 3' end of of poly chain, but chain must have 3' OH to add more nuc 5' triphosphate (dntp) is used and attacked first
Design chain terminating inhibitor
Cant be like dntp bc of side fx Make a dideoxy!!! Resemble substrate
DNA replication basics
1 each strand copy just once 2 time and location or initiation and replication very carefully controlled 3 rep must begin at an origin euk many prok 1 4 bidirectional with rep fork 5 2 strands rep differently 6 DNA pols need primer at least 20 nuc 7 many proteins help 8 low error rate proofreading by dna pol
Draw nucleotide linkage condensation mechansm between 3'oh and 5' po4
Why is there a leading and lagging strand?
DNA polymerase can only add nucleotides in one direction (5' to 3') No adding to 5' end
What proteins are needed for initiation?
1 initation protein 2 helicase 3 ssb 4 primase 5 clamp loading complex 6 sliding clamp 7 DNA pol III (prok only)
Why proteins are need for elongation?
1 DNA pol III 2 DNA pol I 3 DNA ligase 4 topisomerase
Why does helicase need atp?
Has to unwind dna by breaking a lot of h bonds (Hexamer)
SSB
Binds to strands to prevent reanealing and unwanted base pairing, only contacting bb
Clamp loader
Couples with atp to load sliding clamp on dna
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