GENE EXPRESSION

Two steps are required Transcription The synthesis of mRNA uses the gene on the DNA molecule as a template This happens in the nucleus of eukaryotes Translation The synthesis of a polypeptide chain using the genetic code on the mRNA molecule as its guide. © 2010 Paul Billiet ODWS

RIBONUCLEIC ACID (RNA) Found all over the cell (nucleus, mitochondria, chloroplasts, ribosomes and the soluble part of the cytoplasm). © 2010 Paul Billiet ODWS

Types Messenger RNA (mRNA) <5% Ribosomal RNA (rRNA) Up to 80% Transfer RNA (tRNA) About 15% In eukaryotes small nuclear ribonucleoproteins (snRNP). © 2010 Paul Billiet ODWS

Structural characteristics of RNA molecules Single polynucleotide strand which may be looped or coiled (not a double helix) Sugar Ribose (not deoxyribose) Bases used: Adenine, Guanine, Cytosine and Uracil (not Thymine). © 2010 Paul Billiet ODWS

mRNA A long molecule 1 million Daltons Ephemeral Difficult to isolate mRNA provides the plan for the polypeptide chain © 2010 Paul Billiet ODWS

rRNA Coiled Two subunits: a long molecule 1 million Daltons a short molecule 42 000 Daltons Fairly stable Found in ribosomes Made as subunits in the nucleolus rRNA provides the platform for protein synthesis © 2010 Paul Billiet ODWS

tRNA Short molecule about 25 000 Daltons Soluble At least 61 different forms each has a specific anticodon as part of its structure. tRNA “translates” the message on the mRNA into a polypeptide chain © 2010 Paul Billiet ODWS

Transcription: The synthesis of a strand of mRNA (and other RNAs) Uses an enzyme RNA polymerase Proceeds in the same direction as replication (5’ to 3’) Forms a complementary strand of mRNA It begins at a promotor site which signals the beginning of gene is not much further down the molecule (about 20 to 30 nucleotides) After the end of the gene is reached there is a terminator sequence that tells RNA polymerase to stop transcribing NB Terminator sequence ≠ terminator codon. © 2010 Paul Billiet ODWS

Editing the mRNA In prokaryotes the transcribed mRNA goes straight to the ribosomes in the cytoplasm In eukaryotes the freshly transcribed mRNA in the nucleus is about 5000 nucleotides long When the same mRNA is used for translation at the ribosome it is only 1000 nucleotides long The mRNA has been edited The parts which are kept for gene expression are called EXONS (exons = expressed) The parts which are edited out (by snRNP molecules) are called INTRONS. © 2010 Paul Billiet ODWS

Transcription plan Nucleus Gene DNA Transcription messenger RNA © 2010 Paul Billiet ODWS

Translation plan Complete protein Polypeptide chain TRANSLATION Ribosomes Stop codon Start codon © 2010 Paul Billiet ODWS

Translation Location: The ribosomes in the cytoplasm that provide the environment for translation The genetic code is brought by the mRNA molecule. © 2010 Paul Billiet ODWS

What is the genetic code? The genetic code consists of the sequence of bases found along the mRNA molecule There are only four letters to this code (A, G, C and U) The code needs to be complex enough to represent 20 different amino acids used to build proteins. © 2010 Paul Billiet ODWS

How many combinations? If one base represented one amino acid this would only be able to produce 4 different combinations. (A, C, G and U) If pairs of bases represented each amino acid this would only be able to produce 4 x 4 = 16 combinations. (AA, AC, AG, AU, CA, CC, CG, CU etc) If triplets of bases represented each amino acid, this would be able to produce 4 x 4 x 4 = 64 combinations This is enough combinations to code for the 20 amino acids but is the code actually made of triplets? © 2010 Paul Billiet ODWS

Nature is logical! Over 10 years biochemists synthesised bits of mRNA with different combinations Then they used them to synthesise polypeptides The results proved the logical answer was correct The genetic code is made of triplets of bases called codons. © 2010 Paul Billiet ODWS

The Central Dogma Proposed by Francis Crick 1958 DNA holds the coded hereditary information in the nucleus This code is expressed at the ribosome during protein synthesis in the cytoplasm The protein produced by the genetic information is what is influenced by natural selection If a protein is modified it cannot influence the gene that codes for it Therefore there is one way flow of information: DNARNAProtein © 2010 Paul Billiet ODWS

An important discovery Retro viruses (e.g. HIV) carry RNA as their genetic information When they invade their host cell they convert their RNA into a DNA copy using reverse transcriptase Image Credit: Reverse transcriptase Thus the central dogma is modified: DNA↔RNAProtein This has helped to explain an important paradox in the evolution of life.

The paradox of DNA DNA is a very stable molecule It is a good medium for storing genetic material but… DNA can do nothing for itself It requires enzymes for replication It requires enzymes for gene expression The information in DNA is required to synthesise enzymes (proteins) but enzymes are require to make DNA function Which came first in the origin of life DNA or enzymes? © 2010 Paul Billiet ODWS

RIBOZYMES: Both genetic and catalytic Certain forms of RNA have catalytic properties RIBOZYMES Ribosomes and snRNPs are ribozymes RNA could have been the first genetic information synthesizing proteins… …and at the same time a biocatalyst Reverse transcriptase provides the possibility of producing DNA copies from RNA © 2010 Paul Billiet ODWS

The ribosome a ribozyme Image Credit: Ribosome