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Lecture 3. Nucleic acids and enzymes. The nuts and bolts. Most by David Tscharke @ RSB. Lecture overview. How to ‘ read ’ DNA Different types of nucleic acids Cutting DNA by restriction enzymes Synthetic DNA Enzymes used in molecular biology - PowerPoint PPT Presentation
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Nucleic acids and enzymes
The nuts and bolts
Lecture 3
Most by David Tscharke @ RSB
Lecture overview
- How to ‘read’ DNA
- Different types of nucleic acids
- Cutting DNA by restriction enzymes
- Synthetic DNA
- Enzymes used in molecular biology
- polymerases, nucleases, ligase, phosphatases, kinases
DNA is a polymer of 2 long chain polymers…
Modified fromLodish 5th, 4-3 (b)
A C T GP mP m P m P m
CAGTPm PmPmPm
5’ 3’5’3’
… but the chemistry is easy to forget!cattatgataagtacccttatattcacaaatatgatggtgatgagcgacaatattctattactgcagagggaaaatgctataaaggaataaaatatgaaataagtatgatcaacgatgatactctattgagaaaacatactcttaaaattggatctacttatatatttgatcgtcatggacatagtaatacatattattcaaaatatgatttttaaaaatttaaaatatattatcacttcagtgacagtagtcaaataacaaacaacaccatgagatatattataattctcgcagttttgttcattaatagtatacacgctaaaataactagttataagtttgaatccgtcaattttgattccaaaattgaatggactggggatggtctatacaatatatcccttaaaaattatggcatcaagacgtggcaaacaatgtatacaaatgtaccagaaggaacatacgacatatccgcatttccaaagaatgatttcgtatctttctgggttaaatttgaacaaggcgattataaagtggaagagtattgtacgggactatgcgtcgaagtaaaaattggaccaccgactgtaacattgactgaatacgacgaccatatcaatttgtacatcgagcatccgtatgctactagaggtagcaaaaagattcctatttacaaacgcggtgacatgtgtgatatctacttgttgtatacggctaacttcacattcggagattctaaagaaccagtaccatatgatatcgatgactacgattgcacgtctacaggttgcagcatagactttgtcacaacagaaaaagtgtgcgtgacagcacagggagccacagaagggtttctcgaaaaaattactccatggagttcgaaagtatgtctgacacctaaaaagagtgtatatacatgcgcaattagatccaaagaagatgttcccaatttcaaggacaaaatggccagagttatcaagagaaaatttaataaacagtctcaatcttatttaactaaatttctcggtagcacatcaaatgatgttaccacttttcttagcatgcttaacttgactaaatattcataactaatttttattaatgatacaaaaacgaaataaaactgcatattatacactggttaacgcccttataggctctaaccattttcaagatgaggtccctgattatagtccttctgttcccctctatcatctactccatgtctattagacaatgtgagaagactgaagaggaaacatggggattgaaaatagggttgtgtataattgccaaagatttctatcccgaaagaactgattgcagtgttcatctcccaactgcaagtgaaggattgataactgaaggcaatggattcagggatatacgaaacaccgataaattataaaaaaagcaatgtgtccgctgtttccgttaataatactattttcgtaactggcggattattcataaataactctaatagcacgatcgtggttaacaatatggaaaaacttgacatttataaagacaaacaatggtcgattatagaaatgcctatggctagggtatatcacggcatcgactcgacatttggaatgttatattttgccggaggtctatccgttaccgaacaatatggtaa
5 million bp in E. coli – 0.5 million characters in average book
Working with DNA sequences
1 AAAAATTATT GATGTCTACA CATCCTTTTG TAATTGACAT CTATATATCC 51 TTTTGTATAA TCAACTCTAA TCACTTTAAC TTTTACAGTT TTCCCTACCA 101 GTTTATCCCT ATATTCAACA TATCTATCCA TATGCATCTT AACACTCTCT 151 GCCAAGATAG CTTCAAAGTG AGGATAGTCA AAAAGATAAA
Sequence gets written like this:
Questions:
-Is this DNA or RNA?
-Which is the 5’ end?
-Do you need to mark the 5’ end?
Working with DNA sequences 2
Sequence gets written like this:
AAACATTATT GATGTCTACATTTGTAATAA CTACAGATGT3’ 5’
5’ 3’
Question:
-What is the sequence of the complementary strand?
A)TTTGTAATAA CTACAGATGT
B)TGTAGACATC AATAATGTTT
Where do you look if you want to predict the amino acid sequence of the protein?
Working with DNA sequences SUMMARYDNA sequence is often written as a single strand
- but don’t forget the other one
DNA is ALWAYS written 5’ to 3’- except the bottom strand if both strands are written
In CHEM2208, you will ALWAYS lose marks for writing DNA 3’ -> 5’ even if it is labelled
You can predict protein sequence from DNA sequence- proteins may be encoded by the complementary strand- remember to read 5’ -> 3’
Sense and antisenseThe sense strand of a gene has the SAME nucleotide sequence as the mRNA-the antisense strand is complementary to the sense strand
The sense strand of one gene may be the antisense strand of another-Overlapping genes are common in E. coli
5’ AAACATTATTGGTGTCTACA 3’3’ TTTGTAATAACCACAGATGT 5’
Triplets of gene A
Triplets of gene B
How does E. coli identify the reading framesBy a Shine-Dalgarno sequence 10-12 nucleotides prior the start codon
John Shine Lynn Dalgarno
Ribosome binding site (Shine-Dalgarno)Ribosome binding site (RBS)-About 10 nucleotides prior to AUG start codon-Complementary to 16S rRNA of the ribosome-Promotes binding of the ribosome to the mRNA-E. coli consensus RBS is AGGAGGU
Ribosome with mRNA
Shine-Dalgarno helixinvolving 16S RNA
Showing the rRNA of the 30S subunitafter stripping most proteins
Several start codons are possible in E. coliThe classical start codon is AUG (or ATG in DNA; also codes for methionine)
Start codons in E. coli:ATG (83%)GTG (14%)TTG (3%)ATT and CTG (rare)
A start codon always gets translated as methionine even if the codon normally encodes a different amino acid-A separate tRNA is used for initiation
Types of nucleic acid
DNA-genomic -plasmids-PCR products and cDNAs-chemically synthesised DNA (oligonucleotides)
RNA-messenger (mRNA)-transfer (tRNA)-ribosomal (rRNA)-ribozymes-virus genomes
Messenger RNA
As little as 2 – 5 % of total cellular RNA by weight
Lengths from 100s to 1000s of bps
- can be extracted intact
Unstable (all forms of RNA)
- largely due to the ubiquitous presence of RNases
Convert to cDNA for sequencing and expression
Genomic DNA
Genomic DNABacterial DNA does not have nucleosomes
Protein HU -stabilizes left-handed and right-handed supercoiling-there is less of it and its structure is completely different from nucleosomes
Macvanin and Adhya, BBA 2012
Genomic DNA
In your tube after extraction
Conditions before extraction affect integrity
Too much shearing during extraction (e.g. vigorous stirring) leads to breaks
Plasmid DNAExtrachromosomal (or episomal) circles of DNAFound in bacteria and ‘lower’ eukaryotes-yeast has them, mammals do not
Replication is independent of genome
Relatively small-Thousands rather than millions of base pairs-remains intact after extraction
Exists in various states of supercoiling- supercoiled, closed relaxed circular,
open circular, linear
Small plasmids are easy to manipulate
Gene
Segment of DNA coding for protein or (functional) RNA
Restriction endonucleasesAKA ‘Restriction enzymes’
Recognise dsDNA sequences 4-8 base pairs long
Cut both strands
Restriction/modification systems provide anti-viral immunityto bacteria
Type II (and IIS) used in molecular biology-Cutting and modification are done by different enzymes-Cut the DNA within (type II) or outside (IIS) the recognition site
Cut ends have 5’-phosphate groups
Type II Restriction enzymes : EcoRI
G A A T T C
C T T A A G
site of cleavage2-fold axis of symmetry (rotational)
This kind of sequence is called a palindrome!It is different to a palindrome in language
The phosphate ester groups stay on the 5’ ends
SmaI
EcoRI
Restriction cutting can give different ends
5’ GAATTC G AATTC
3’ CTTAAG CTTAA G
5’ CCCGGG CCC GGG
3’ GGGCCC GGG CCC
5’ CTGCAG CTGCA G
3’ GACGTC G ACGTC
5’ overhang5’ protruding5’ sticky
3’ overhang3’ protruding3’ sticky
blunt
PstI
Ends can be re-joined by ligaseif the 5’phopshate is intact (later lecture)
IsoscizomersEnzymes with the same recognition sequence- May not cut in the same place
▼GATC CTAG▲
C▼CCGG GG GGCC▲C Sma I
CCC▼GGGGGG▲CCC Xma I
G CATG▼CC▲GTAC G Sph I & Bbu I
Sau3A I,Mbo I & Nde II
G*A ▼T CC T▲*AG Dpn I
Question timeHow are restriction enzymes named?
HindIII
EcoRI
PstI
BamHI
BglII
SexAI
3rd from Haemophilus influenzae, d
1st from Eschericia coli, R
2nd from Bacillus globigii
1st from Bacillus amyloliquefaciens, H
1st from Providencia stuartii
1st from Streptomyces exfoliatus
How many are there, what do they cost?
Company number price range
Promega 96 $1 - $240 / 100u
New England Biolabs
225 50c - $60 / 100u
Roche Applied Science
112 50c - $260 / 100u
Invitrogen 47 $1 - $100 / 100u
Biotech shopping list• Polymerases
• Nucleases– Endo- and exo-
• Ligases
• Kinases
• Phosphatases
Enzymes
Need the right conditions of salt and temperature
Some need extras, such as ATP or metal ions (Mg2+)
Some are stable, most are destroyed by heat– Or even leaving out of the freezer for a few hours
Most are stored in buffer containing glycerol– Keeps them liquid at -20 ºC– Saves them from freeze/thaw cycles– BUT limits the amount you can use
http://www.vivo.colostate.edu/hbooks/genetics/biotech/enzymes/index.html
If DNA is the building material, enzymes are the tools
Chemically synthesised DNA
Oligonucleotides or ‘oligos’
Used extensively in molecular biology- primers for PCR- synthetic genes
ssDNA
Lack 5’ phosphate- unless you ask
Chemical synthesis of DNA
Silicabead
Silicabead
Silicabead
Deblocking
Activation& Coupling
Capping(unreacted)
Oxidation
Glick pp91-95
Errors add up
0
20
40
60
80
100
0 20 40 60 80 100
Length of desired product (bp)
overallyield
of desiredproduct
(%)90%95%98%99%
The relationship is exponential
couplingefficiency
Chemical DNA synthesis
Can make any length- ssDNA or dsDNA - 5’ phosphate costs extra
Around $0.5 a base for most applications
Up to 30-mer usually OK to have unpurified for many(but not all) applications
- purification by HPLC or gel methods (for correct size)
May get occasional base substitutions- even if purified by gel, can have some errors (if not
checked by sequencing)
Summary of nucleic acid section
Need to remember your chemistry- ssDNA lacking a 5’ phosphate
Different nucleic acids have different properties- can be the basis for separations / purification
DNA can be made chemically- only efficient for short bits (<100 bp)- always have some copies with errors
Molecular biology shopping list
• Polymerases
• Nucleases– Endo- and exo-
• Ligases
• Kinases
• Phosphatases
Polymerases
All add nucleotides in a 5’ -> 3’ direction
Examples:– Klenow fragment, Taq and Pfu polymerases
- DNA-dependent DNA polymerase
– Reverse transcriptase- RNA-dependent DNA polymerase
– T7 and SP6 phage RNA polymerases- RNA-dependent RNA polymerase
Many have extra functions
- Some proof-read (3’ -> 5’ exonuclease activity)- Some are also terminal transferases
All polymerases need ‘priming’
?
DNApol
3’ 5’
5’ 3’
!
Also need the building blocks…dATP, dCTP, dGTP and dTTP (often called dNTPs)
Primers in molecular biology
Primers direct the start of DNA synthesis
Great… as long as you know the sequence
Synthetic oligonucleotides most commonly used
Must get conditions right for primer hybridisation AND polymerase activity
- primers must have the right length for desired specificity
Endonucleases and Exonucleases
Cleave one or more phosphodiester bonds
Restriction endonucleases are some of the most important tools in biotechnology
– Cleave both strands of dsDNA at a specific sequence– We’re going there next lecture…
In general they are the enemy, when controlled are very useful
Endo
ExoExo
5’
3’
3’
5’
Ligase
Ligase repairs broken phoshodiester bonds
– Uses ATP (one for each bond repaired)
– Most common enzyme for joining DNA in vitro
– Needs 5’-phosphate
Ligase
from Lodish Fig 9-11
Phosphatases / kinases
3’ 5’
5’ 3’P
P
3’ 5’
5’ 3’
polynucleotide kinasealkaline phosphatase
ATP
ADP
ATP
Enzymes Summary
Make DNA or RNA (biologically, in vitro)– polymerases
Cut nucleic acid or degrade it– nucleases
Join DNA– ligases
Mess with the ends of DNA to alter joining– kinase / phosphatase
Naturally ocurring enzymes supply all the tools to:
Never assume: 1) they only do exactly what you want2) they do it with 100% efficiency