BCM208 Metabolic Biochemistry Topic 7: Gene metabolism and Expression

Learning Objectives Describe the structure of DNA Understand the processes involved in DNA, RNA and protein synthesis Understand the process of gene expression Understand how genes are regulated Understand the basic concept of recombinant DNA technology Understand how plants are transformed using Agrobacterium

The flow of genetic information

Structure of DNA

Structure of DNA (cont.)

DNA Replication

DNA Replication (cont.)

DNA replication

Transciption

Transcription

RNA processing

Decoding Genetic Information: RNA and Protein

Translation

Translation (cont.)

Regulating gene expression

Regulation of mRNA transcription in Bacteria

Negative regulation

Positive regulation

Positive and negative regulation

A repressible operon

An inducible operon

lac operon (cont.)

Regulation of mRNA transcription in Eukaryotes Most active eukaryotic cells transcribe a common (basal) set of structural genes that maintain routine (household) cellular functions Cells express other specialised genes which give the cells there unique properties

Regulation of mRNA transcription in Eukaryotes (cont.) A number of diverse, highly specific processes that activate or repress transcription in eukaryotic cells Generally transcription is mediated by proteins that are collectively classified as transcription factors

Regulation of mRNA transcription in Eukaryotes (cont.) Transcription factors bind to DNA sequences (often called boxes) There are some general regulatory sequences, however, most genes have their own set of response elements

Common eukaryotic regulatory sequences TATA box: (or Hogness box) 8 nucleotides that includes a TATA sequence (-25 bp) “cat” box: CCAAT (-75 bp) GC box: a sequence of repeated GC nucleotides (-90 bp)

Eukaryotic gene regulation is complex!!!

DNA cloning Why clone DNA? : To allow large scale amplification of identical molecules What for?: –Further analysis eg DNA sequencing –Expression of a gene –Insertion into transgenic organism

DNA cloning (cont.)

Enzymes used in recombinant DNA technology

Restriction enzymes Cleave DNA at specific sequences Isolated from bacteria (eg Eco RI isolated from E. coli) Natural role: cleave invading DNA viruses

Recognition sequences

Sticky vs blunt ends

Plasmid cloning vector (pBR322)

Polylinker

DNA libraries Obtain DNA fragments –cDNA –Genomic DNA digested with RE Digest plasmid DNA with RE Ligate DNA fragments to digested plasmid Transform E.coli: each cell contains different DNA fragment Generate colonies for individual cells

Applications of DNA libraries Looking for genes expressed in particular tissues (cDNA) Identifying genes using southern hybridisation (requires probe with complementary sequence eg derived from a similar gene from a different species) Western analysis can also be used to screen libraries (requires gene to be expressed in bacteria)

Electrophoresis Migration of DNA, RNA or protein through a matrix Molecules move due to charge: migrate toward +ve electrode (due to negatively charged phosphate groups in nucleic acids) Smaller molecules are able to migrate more rapidly that larger molecules Migration monitored by a visible dye

Agarose gel Electrophoresis Agarose: polysaccharide from seaweed Used to separate DNA and RNA molecules above 300 bp Prepared by dissolving agarose powder (by heating in microwave) in running buffer (usually Tris- Borate EDTA buffer or TBE) Agarose concentration range: 0.8% - 2% Higher concentration resolve lower molecular weight molecules more effectively

Visualisation of DNA and RNA Ethidium bromide most commonly used for nucleic acid visualisation Binds to nucleic acids and fluoresces when exposed to UV light Mutagen

Acrylamide gel electrophoresis Used for Proteins and DNA molecules smaller that 300 bp (eg DNA sequencing) Higher resolution Pore size determined by acrylamide concentration Acrylamide monomer is a neurotoxin

Protein electrophoresis Protein often treated with sodium dodecyl sulphate (SDS) to interrupt inter-molecular bonding so that molecules run based on molecular weight

Expression of eukaryotic genes in prokaryotic systems Eukaryotic genes contain introns To express eukaryotic genes in prokaryotic cells, introns need to be removed This can be done by generating DNA copies of mRNA (which have had introns spliced out) This DNA copy of mRNA is called complementary DNA (or cDNA)

Plant transformation Microprojectile bombardment Agrobacterium

Microprojectile Bombardment

Plant transformation with Agrobacterium tumefaciens

Plant regeneration Fig 29-19

Insect resistant transgenic plants Fig

Herbicide resistant transgenic plants Fig 29-22