Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the...

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Lecture Notes for

Chapter 3 From Genes to Proteins

Essential Biochemistry Third Edition

Charlotte W. Pratt | Kathleen Cornely

KEY CONCEPTS: Section 3-1

•  DNA and RNA are polymers of nucleotides, each of which consists of a: – Purine or pyrimidine base

– Deoxyribose or ribose

– Phosphate

DNA and RNA have four nitrogenous bases.

•  Purines: 2 examples •  Pyrimidines: 3 examples •  Notice the similarities in structure. •  Notice differences in numbering around ring.

Adenine and guanine are purines in both DNA and RNA.

Learn how to draw purines. Adenine, A

•  Draw ring system •  Two more steps

– Add amino group (C6) – Fill in bonds

Guanine, G •  Draw ring system •  Three more steps

– Add amino group (C2) – Add oxo group (C6) – Fill in bonds

Cytosine and thymine are pyrimidines found in DNA.

Cytosine and uracil are pyrimidines found in RNA.

Learn how to draw pyrimidines. Cytosine, C

•  Draw ring system

•  Two more steps –  Add amino group (C4) –  Fill in bonds

Uracil, U and Thymine, T •  Draw ring system

•  Two, three more steps –  Add oxo group(C4) –  Add H on N3 –  Add methyl group (C5)

for thymine

DNA and RNA contain sugar groups. Found in RNA Found in DNA

Notice: Ring numbering

on the sugars contain primes!

Phosphates can attach to the sugar at C3’ and/or C5’.

Monophosphates are shown in examples here. There can be up to 3 phosphates at a given terminus.

Nucleoside/nucleotide nomenclature is distinct from that of bases.

•  Nucleoside = base + sugar

•  Nucleotide = base + sugar + phosphate

Nomenclature Summary Base Nucleoside Nucleotide (example using monophosphates) Adenine Adenosine Adenosine Monophosphate Guanine Guanosine Guanosine Monophosphate Thymine Thymidine Thymidine Monophosphate Cytosine Cytidine Cytidine Monophosphate Uracil Uridine Uridine Monophosphate

Some nucleotides have functions other than encoding genetic information.

Nucleotides can be fused with vitamins to form molecules that aid in enzyme-catalyzed reactions.

(vitamin)

How do nucleotides link?

•  Via sugar in phosphodiester backbone

•  Read sequence of bases from 5’à 3’

•  Nucleotides are connected via phosphodiester bonds.

Bases form specific hydrogen bonds.

A and T form 2 H-bonds.

G and C form 3 H-bonds.

Base pair width is similar within a given conformation

Chargaff’s rule gives a clue to base pairing.

•  Chargaff’s rule: the amount of A+G = C+T.

•  Base pairs contain a purine and a pyrimidine.

•  A base pairs with T or U; G base pairs with C.

•  A DNA molecule contains two antiparallel strands that wind around each other to form a double helix in which A and T bases in opposite strands, and C and G bases in opposite strands, pair through hydrogen bonding.

DNA forms a double helix.

Ball-and-stick representation

Space-filling representation with phosphate

backbone emphasized in

white.

Strands are antiparallel.

What drives DNA to form a double helical shape in 3D?

Water plays a role in dictating biomolecular structure via the

hydrophobic effect.

The hydrophobic effect is the phenomenon by which nonpolar molecules aggregate to avoid contact with

hydrophilic molecules, particularly water.

From Chapter 2

Unfavorable Many H2O molecules

are ordered around the nonpolar molecules.

Preferred Fewer H2O molecules

are ordered around the nonpolar molecules.

As H2O molecules are freed, entropy

of the system increases!

Axial View of DNA

Base pairs form the core of the double helix.

Phosphate backbone forms the periphery.

Double helical conformation of DNA is driven by entropic

forces that induce base stacking.

Viewing 3D Structures of Macromolecules

•  Go to http://www.rcsb.org/pdb/home/home.do

•  Search for a molecule of interest.

•  Select its link (e.g. 355D).

•  Select download files à PDB file (text).

•  Open the file in molecular viewing freeware.

DNA is stabilized by different forces.

•  Predominant force: hydrophobicity, base stacking and entropy

•  Hydrogen bonding (in base pairs) •  Ionic interactions

– Cations (e.g. Mg2+, Na+, K+) – Polyamines

•  Double-stranded nucleic acids are denatured at high temperatures.

•  At lower temperatures, complementary

polynucleotides anneal.

DNA can denature (unfold). •  Native state •  Denatured

•  Abs = 260 nm

•  Tm = melting temperature

DNA can renature (refold, anneal).

DNA’s ability to re-anneal is very important – in nature and in biochemical research!

•  The biological information encoded by a sequence of DNA is: – Transcribed to RNA – Then, translated into the amino acid sequence

of a protein

What does it mean to go from genes to proteins?

•  Genes are sequences of DNA. •  Replication: copying DNA •  Transcription: converting DNA into RNA •  Reverse transcription: converting RNA into DNA •  Translation: making proteins from an RNA

DNA replication is critical to life.

•  In vivo – DNA must be copied in order to sustain life. – Excessive DNA replication can be indicative of

cancer.

•  In vitro – Technology known as the “polymerase chain

reaction” or PCR has revolutionized researchers capability to study nucleic acids and genes.

Transcription of DNA is critical to life.

Cells cannot thrive if transcription is shut down!

Transcription RNA polymerase (not shown here) unwinds and separates dsDNA at the position

where transcription occurs.

Notice that the RNA transcript is complementary to the antisense (noncoding) strand!

There are three fundamental types of RNA.

•  Messenger RNA: encodes for polypeptide sequences

•  Transfer RNA: carries amino acids to ribosome

•  Ribosomal RNA: aids in polypeptide synthesis

tRNA is single-stranded and forms unique conformations.

Translation results in protein synthesis.

Translation occurs in the ribosome, which contains rRNA and many other proteins.

In translation, tRNA carries amino acids to the ribosome and

binds to its complement in the mRNA template.

Amino acids are dictated by the Genetic Code.

The genetic code is used to translate mRNA into an amino acid sequence.

Read mRNA sequence in the Genetic Code

by the position of 3 nucleotides.

Notice the redundancy!

•  The genomes of different species vary in size and number of genes.

•  Genes can be identified by their nucleotide sequences.

•  Analysis of genetic data can provide information about gene function and risk of disease.

Gene size is roughly correlated with organismal complexity.

•  A DNA molecule can be sequenced or amplified by using DNA polymerase to make a copy of a template strand.

•  Linking together DNA fragments produces recombinant DNA molecules that can be used: – To study gene function – To express genes in host organisms – To engineer genes for commercial and

therapeutic purposes

DNA can be sequenced via the Sanger method.

ddNTPs lack a 3’-OH group

No more NTP’s

can attach.

Dideoxy DNA Sequencing – Sanger Method

DNA can be amplified or copied using DNA polymerase.

•  A process called the “Polymerase Chain Reaction” or PCR can be used to make billions of copies of DNA efficiently and accurately.

•  PCR was developed by Kary Mullis, who won the 1993 Nobel Prize in Chemistry for his discovery.

What is required to make PCR work? •  Heat-stable DNA polymerase (e.g., “Taq”

DNA polymerase)

•  Primers (DNA oligonucleotides)

•  Deoxynucleotide triphosphates

– dATP, dGTP, dCTP, dTTP

•  DNA template

•  Buffer containing Mg2+, among others

Also required:

Thermal Cycler – a device that heats and cools samples with speed, precision, and

reproducibility.

PCR repeats three chemical reactions.

•  Step 1: Denaturation 92–95°C – dsDNA separates at high temp to form ssDNA.

•  Step 2: Annealing ~55°C – Primers can base pair to ssDNA.

•  Step 3: Extension 72°C

– Optimal temp for heat-stable DNA polymerases to work

PCR is conducted over many cycles until millions of

copies are produced.

Restriction enzymes’ role in nature is to aid in defense for bacteria.

•  Bacteria methylate their own DNA

•  Bacteriophages (viruses that infect bacteria) have unmethylated DNA.

•  Bacterial restriction enzymes can recognize and excise viral DNA.

Type II restriction enzymes are used to selectively cut DNA.

Type Activity Cleavage Site I Endonuclease & ≥ 1000 bp from

Methylase recognition sequence II Endonuclease Within or near

recognition sequence III Endonuclease & ~24-26 bp from

Methylase recognition sequence

EcoRV 5’-G-A-T-A-T-C-3’ 3’-C-T-A-T-A-G-5’

EcoRI 5’-G-A-A-T-T-C-3’ 3’-C-T-T-A-A-G-5’

Characteristics of Type II Restriction Enzymes

•  Cleave 4-8 bp segment of dsDNA

•  Palindromic recognition sequences – “Madam, I’m Adam” – “Race Car”

Remember: Always read from

5' à 3'

Axis of symmetry

Type II Restriction enzymes form blunt or sticky ends.

•  EcoRI recognizes 5'-GAATTC-3'. •  Complimentary sequence is implied. •  EcoRI cuts both strands after the 5'-G. •  Each fragment has sticky ends.

5' 5' 3' 3'

Type II Restriction enzymes form blunt or sticky ends.

•  EcoRV recognizes 5'-GATATC-3'. •  Complimentary sequence is implied. •  EcoRV cuts both strands after the 5'-T. •  Each fragment has blunt ends.

DNA fragment sizes can be quantified from gel images.

•  Gel electrophoresis is a method of separating DNA fragments by size.

•  Gel is made of agarose.

•  DNA bands can be stained with chemicals such as ethidium bromide for fluorescent detection.

What is a DNA Plasmid? •  Double-stranded, extra-chromosomal DNA •  Size: 1–200 kbases •  Covalently closed, circular, superhelical •  Bacterial •  Dependent on host cell’s proteins for replication

and transcription machinery

Site-Directed Mutagenesis

A method of introducing a single amino acid change in a protein as a result of a mutation at a specific site in the DNA.

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