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