PROTEIN SYNTHESIS An individual’s characteristics are determined by their DNA. The DNA code determines which proteins are made. The sequence of bases in the DNA determines the sequence of amino acids in the protein. This is known as the GENETIC CODE.

THE GENETIC CODE (and terms to learn) CODON 3 nucleotides code for one amino acid in the protein. Each triplet in mRNA is known as a CODON (it is called a DNA triplet when the code is in the DNA) The code is UNIVERSAL Meaning, the same nucleotide bases are found in all organisms (A,G,T,C,U) Also, the same codons (triplet code), code for the same types of amino acids in ALL organisms The code is REDUNDANT More than one codon can code for a particular amino acid. (but no codon codes for more than one type of amino acid)

This table shows codons on mRNA – some tables show the DNA code

Reading the Codon Table (some examples) UUU codes for the amino acid, phenylalanine CCA codes for the amino acid, proline The codon, CCG also codes for proline this demonstrates REDUNDANCY in the genetic code Frogs, plants, bacteria etc have the same codons which code for the same amino acids this demonstrates that the DNA code is UNIVERSAL

START and STOP Codons START codon AUG (remember this code!!) Signals the beginning of transcription (the beginning of code for the protein) STOP codons UAA, UAG, UGA Signals the end of transcription (the end of the code for the protein)

Protein Synthesis Occurs in 2 main stages: Transcription (in the nucleus) DNA transcribed (copied) into pre-mRNA Pre-mRNA has introns removed and exons recombined in alternative ways (alternative splicing) to produce the final mRNA 2. Translation (in the cytoplasm, at ribosomes) mRNA codons are translated into the amino acid sequence (tRNA’s carry the specific amino acids required)

Protein Synthesis Summary Pre-mRNA then undergoes alternative splicing to produce mRNA (which also has a poly-A tail and 5’ cap added)

TRANSCRIPTION

An Overview of Gene Structure Coding Region DNA sequence that will be transcribed from the template strand. 5’ 3’ 5’ STOP Regulatory region START 3’ Promoter region Terminator region

U C A C U U G U A C A G G A A U U A G T A C G G C T A A C A T A C A A T C G U HELICASE unwinds and unzips the relevant part of the DNA helix. RNA POLYMERASE attaches to the DNA (at the PROMOTOR SEQUENCE). One DNA strand acts as the template (SENSE STRAND), the other is redundant (ANTISENSE STRAND). As RNA POLYMERASE moves along the template strand (reading it from 3’ to 5’ direction), ribonucleotides are assembled in a precise order due to complementary base pairing.

U C A C U U G U A C A G G A A U U A G T A C G G C T A A C A T A C A A T C G U HELICASE unwinds and unzips the relevant part of the DNA helix. RNA POLYMERASE attaches to the DNA (at the PROMOTOR SEQUENCE). One DNA strand acts as the template (SENSE STRAND), the other is redundant (ANTISENSE STRAND). As RNA POLYMERASE moves along the template strand (reading it from 3’ to 5’ direction), ribonucleotides are assembled in a precise order due to complementary base pairing.

U C A C U U G U A C A G G A A U A U G T A C G G C T A A C A T A C A A T C G U HELICASE unwinds and unzips the relevant part of the DNA helix. RNA POLYMERASE attaches to the DNA (at the PROMOTOR SEQUENCE). One DNA strand acts as the template (SENSE STRAND), the other is redundant (ANTISENSE STRAND). As RNA POLYMERASE moves along the template strand (reading it from 3’ to 5’ direction), ribonucleotides are assembled in a precise order due to complementary base pairing.

U C A C U U G U A C A G A A U A U G G T A C G G C T A A C A T A C A A T C G U HELICASE unwinds and unzips the relevant part of the DNA helix. RNA POLYMERASE attaches to the DNA (at the PROMOTOR SEQUENCE). One DNA strand acts as the template (SENSE STRAND), the other is redundant (ANTISENSE STRAND). As RNA POLYMERASE moves along the template strand (reading it from 3’ to 5’ direction), ribonucleotides are assembled in a precise order due to complementary base pairing.

U C A U U G U A C A G A A U A U G C G T A C G G C T A A C A T A C A A T C G U HELICASE unwinds and unzips the relevant part of the DNA helix. RNA POLYMERASE attaches to the DNA (at the PROMOTOR SEQUENCE). One DNA strand acts as the template (SENSE STRAND), the other is redundant (ANTISENSE STRAND). As RNA POLYMERASE moves along the template strand (reading it from 3’ to 5’ direction), ribonucleotides are assembled in a precise order due to complementary base pairing.

U C A U U G U A A G A A U A U G C C G T A C G G C T A A C A T A C A A T C G U HELICASE unwinds and unzips the relevant part of the DNA helix. RNA POLYMERASE attaches to the DNA (at the PROMOTOR SEQUENCE). One DNA strand acts as the template (SENSE STRAND), the other is redundant (ANTISENSE STRAND). As RNA POLYMERASE moves along the template strand (reading it from 3’ to 5’ direction), ribonucleotides are assembled in a precise order due to complementary base pairing.

U C A U U G U A A A A U A U G C C G G T A C G G C T A A C A T A C A A T C G U HELICASE unwinds and unzips the relevant part of the DNA helix. RNA POLYMERASE attaches to the DNA (at the PROMOTOR SEQUENCE). One DNA strand acts as the template (SENSE STRAND), the other is redundant (ANTISENSE STRAND). As RNA POLYMERASE moves along the template strand (reading it from 3’ to 5’ direction), ribonucleotides are assembled in a precise order due to complementary base pairing.

U C A U U G U A A A U A U G C C G A G T A C G G C T A A C A T A C A A T C G U HELICASE unwinds and unzips the relevant part of the DNA helix. RNA POLYMERASE attaches to the DNA (at the PROMOTOR SEQUENCE). One DNA strand acts as the template (SENSE STRAND), the other is redundant (ANTISENSE STRAND). As RNA POLYMERASE moves along the template strand (reading it from 3’ to 5’ direction), ribonucleotides are assembled in a precise order due to complementary base pairing.

U C A U G U A A A U A U G C C G A U G T A C G G C T A A C A T A C A A T C G U HELICASE unwinds and unzips the relevant part of the DNA helix. RNA POLYMERASE attaches to the DNA (at the PROMOTOR SEQUENCE). One DNA strand acts as the template (SENSE STRAND), the other is redundant (ANTISENSE STRAND). As RNA POLYMERASE moves along the template strand (reading it from 3’ to 5’ direction), ribonucleotides are assembled in a precise order due to complementary base pairing.

U C A U G A A A U A U G C C G A U U G T A C G G C T A A C A T A C A A T C G U HELICASE unwinds and unzips the relevant part of the DNA helix. RNA POLYMERASE attaches to the DNA (at the PROMOTOR SEQUENCE). One DNA strand acts as the template (SENSE STRAND), the other is redundant (ANTISENSE STRAND). As RNA POLYMERASE moves along the template strand (reading it from 3’ to 5’ direction), ribonucleotides are assembled in a precise order due to complementary base pairing.

U C A U G A A A U A U G C C G A U U G T A C G G C T A A C A T A C A A T C G U HELICASE unwinds and unzips the relevant part of the DNA helix. RNA POLYMERASE attaches to the DNA (at the PROMOTOR SEQUENCE). One DNA strand acts as the template (SENSE STRAND), the other is redundant (ANTISENSE STRAND). As RNA POLYMERASE moves along the template strand (reading it from 3’ to 5’ direction), ribonucleotides are assembled in a precise order due to complementary base pairing.

U C A G A A A U A U G C C G A U U G U T A C G G C T A A C A T A C A A T C G U HELICASE unwinds and unzips the relevant part of the DNA helix. RNA POLYMERASE attaches to the DNA (at the PROMOTOR SEQUENCE). One DNA strand acts as the template (SENSE STRAND), the other is redundant (ANTISENSE STRAND). As RNA POLYMERASE moves along the template strand (reading it from 3’ to 5’ direction), ribonucleotides are assembled in a precise order due to complementary base pairing.

U C A G A A U A U G C C G A U U G U A T A C G G C T A A C A T A C A A T C G U HELICASE unwinds and unzips the relevant part of the DNA helix. RNA POLYMERASE attaches to the DNA (at the PROMOTOR SEQUENCE). One DNA strand acts as the template (SENSE STRAND), the other is redundant (ANTISENSE STRAND). As RNA POLYMERASE moves along the template strand (reading it from 3’ to 5’ direction), ribonucleotides are assembled in a precise order due to complementary base pairing.

C A G A A U A U G C C G A U U G U A U T A C G G C T A A C A T A C A A T C G U HELICASE unwinds and unzips the relevant part of the DNA helix. RNA POLYMERASE attaches to the DNA (at the PROMOTOR SEQUENCE). One DNA strand acts as the template (SENSE STRAND), the other is redundant (ANTISENSE STRAND). As RNA POLYMERASE moves along the template strand (reading it from 3’ to 5’ direction), ribonucleotides are assembled in a precise order due to complementary base pairing.

C A A A U A U G C C G A U U G U A U G T A C G G C T A A C A T A C A A T C G U HELICASE unwinds and unzips the relevant part of the DNA helix. RNA POLYMERASE attaches to the DNA (at the PROMOTOR SEQUENCE). One DNA strand acts as the template (SENSE STRAND), the other is redundant (ANTISENSE STRAND). As RNA POLYMERASE moves along the template strand (reading it from 3’ to 5’ direction), ribonucleotides are assembled in a precise order due to complementary base pairing.

C A A A U A U G C C G A U U G U A U G U T A C G G C T A A C A T A C A A T C G HELICASE unwinds and unzips the relevant part of the DNA helix. RNA POLYMERASE attaches to the DNA (at the PROMOTOR SEQUENCE). One DNA strand acts as the template (SENSE STRAND), the other is redundant (ANTISENSE STRAND). As RNA POLYMERASE moves along the template strand (reading it from 3’ to 5’ direction), ribonucleotides are assembled in a precise order due to complementary base pairing.

C A A A A U G C C G A U U G U A U G U U T A C G G C T A A C A T A C A A T C G HELICASE unwinds and unzips the relevant part of the DNA helix. RNA POLYMERASE attaches to the DNA (at the PROMOTOR SEQUENCE). One DNA strand acts as the template (SENSE STRAND), the other is redundant (ANTISENSE STRAND). As RNA POLYMERASE moves along the template strand (reading it from 3’ to 5’ direction), ribonucleotides are assembled in a precise order due to complementary base pairing.

C A A A U G C C G A U U G U A U G U U A T A C G G C T A A C A T A C A A T C G HELICASE unwinds and unzips the relevant part of the DNA helix. RNA POLYMERASE attaches to the DNA (at the PROMOTOR SEQUENCE). One DNA strand acts as the template (SENSE STRAND), the other is redundant (ANTISENSE STRAND). As RNA POLYMERASE moves along the template strand (reading it from 3’ to 5’ direction), ribonucleotides are assembled in a precise order due to complementary base pairing.

C A A U A G C T A C G G C T A A C A T A C A A T C HELICASE unwinds and unzips the relevant part of the DNA helix. RNA POLYMERASE attaches to the DNA (at the PROMOTOR SEQUENCE). One DNA strand acts as the template (SENSE STRAND), the other is redundant (ANTISENSE STRAND). As RNA POLYMERASE moves along the template strand (reading it from 3’ to 5’ direction), ribonucleotides are assembled in a precise order due to complementary base pairing.

Pre-mRNA to final mRNA The mRNA is actually pre-mRNA at this stage as it also contains a copy of the introns (non-coding regions) The introns are removed in a process called splicing or editing (also call post-transcription modification) A 5’ cap is also added to allow ribosomes to attach to the mRNA A poly-A tail is added to the 3’ end which helps stabilise the mRNA, guide it through the nuclear pore to the cytoplasm and assist ribosome attachment

Post transcription modification of pre-mRNA

Alternative Splicing The exons can be recombined in different combinations Each combination results in a different final mRNA that codes for a different protein

Alternative Splicing http://highered.mheducation.com/sites/9834092339/student_view0/chapter16/animation_-_exon_shuffling.html

U A G C Fully formed mRNA leaves the nucleus via a nuclear pore.

U A G C Fully formed mRNA leaves the nucleus via a nuclear pore.

U A G C

U A G C

SPECIFIC AMINO ACIDS ARE ATTACHED TO SPECIFIC tRNA’s (each tRNA has it’s own specific 3 nucleotide code, called an anti-codon, which is complimentary to the mRNA’s codon)

THE ANTICODON DETERMINES WHICH SPECIFIC AMINO ACID IS ATTACHED EACH tRNA COMBINES WITH A SPECIFIC AMINO ACID U A C tRNA THE ANTICODON DETERMINES WHICH SPECIFIC AMINO ACID IS ATTACHED

(occurs in the cytosol at ribosomes) TRANSLATION (occurs in the cytosol at ribosomes)

A ribosome binds to the mRNA near the START CODON. U U A C G C U A A C A U G C C G A U U G U A U G U U A G A ribosome binds to the mRNA near the START CODON.

C A U U A G G C U A C A C A U G C C G A U U G U A U G U U A G tRNA with the complementary ANTICODON (UAC) binds to the start codon (AUG) held in place by the large subunit of the ribosome. It brings with it the amino acid methione.

The ribosome now slides along the mRNA to “read” the next codon. U U A G G C U A C A C A U G C C G A U U G U A U G U U A G The ribosome now slides along the mRNA to “read” the next codon.

C A U U A U A C A C G G C A U G C C G A U U G U A U G U U A G A second tRNA now bind to this codon, bringing a second amino acid with it.

A peptide bond is formed between the two amino acids. U U A U A C A C G G C A U G C C G A U U G U A U G U U A G A peptide bond is formed between the two amino acids.

C A U U A C U A A C G G C A U G C C G A U U G U A U G U U A G The tRNA which carried the first amino acid is released but leaves its amino acid behind as a DIPEPTIDE.

The ribosome now slides along the mRNA to “read” the next codon. U U A C U A A C G G C A U G C C G A U U G U A U G U U A G The ribosome now slides along the mRNA to “read” the next codon.

One by one each codon is read as the ribosome moves along the mRNA. U U A C U A A C G G C A U G C C G A U U G U A U G U U A G One by one each codon is read as the ribosome moves along the mRNA.

C A U U A C U A A C G G C A U G C C G A U U G U A U G U U A G Each time the growing polypeptide is linked to the amino acid on the incoming tRNA.

C A U U A C G C A A C U A U G C C G A U U G U A U G U U A G

U A C G C A C A U A C U A U G C C G A U U G U A U G U U A G

U A C G C A C A U A C U A U G C C G A U U G U A U G U U A G

U A C U A G C C A U A C A U G C C G A U U G U A U G U U A G

U A C U A G C C A U A C A U G C C G A U U G U A U G U U A G

U A C U A G C C A U A C A U G C C G A U U G U A U G U U A G

U A C U A U A C G C A C A U G C C G A U U G U A U G U U A G

The polypeptide is complete when the ribosome reaches the STOP codon. U A C U A U A C G C A C STOP ! A U G C C G A U U G U A U G U U A G The polypeptide is complete when the ribosome reaches the STOP codon.

The polypeptide is released. C U A C C A U U A G C A U G C C G A U U G U A U G U U A G The polypeptide is released.

The polypeptide is released. C U A C C A U U A G C A U G C C G A U U G U A U G U U A G The polypeptide is released.

The polypeptide in this form is the primary structure The polypeptide in this form is the primary structure. It will further fold into secondary structures and finally a tertiary structure. It may combine with other polypeptides to produce a quaternary structure.

G C U A The tRNAs are recycled.

A U G C C G A U U G U A U G U U A G The mRNA may be used again in this form, or it may be broken down into nucleotides which can be reassembled to produce a different polypeptide.

The ribosome is free to move along another mRNA.

Ribosomes work in groups so that many slide along an mRNA molecule simultaneously. These groups are called POLYRIBOSOMES. Each ribosome takes about 1 minute to travel along an mRNA molecule.

And post-transcriptional modification (including alternative splicing) Amino acid tRNA Ribosome

Transcription and Translation https://www.youtube.com/watch?v=2zAGAmTkZNY https://www.youtube.com/watch?v=8nQH0GqFn6k

Eukaryote vs Prokaryote DNA protein synthesis No pre-mRNA (no processing, as no introns) and both transcription and translation occur in the cytoplasm

QUIZ What sort of chemical is helicase? Why is DNA double stranded if one strand is redundant? Where in the cell are the ribosomes? What is the start codon? Give the three alternative stop codons. Give the primary structure (sequence of amino acids) of the polypeptide made in this animation. What is the difference between a polypeptide and a protein? What is the advantage of ribosomes operating as polyribosomes? What are the similarities and differences between DNA replication and protein synthesis?

ANSWERS Helicase is an enzyme and therefore also a protein. DNA is double stranded to permit replication. Ribosomes are located in the cytoplasm. The start codon is AUG. The three stop codons are UGA, UAG and UAA. Give the primary structure of the polypeptide is methionine, proline, isoleucine, valine, cysteine. Polypeptides have less than 100 amino acids, protein have more. A protein may consist of several polypeptides. Polysomes increase efficiency, they enable one mRNA molecule to produce many polypetides simultaneously. HINT - Think about the enzymes and nucleotides used, the end product and the location of the process.

Different types of genes Segment of DNA that codes for formation of a protein Structural genes code for structural and/or functional proteins. Regulatory genes are short nucleotide sequences that code for proteins that control the activity of structural genes.

Gene Regulation Each cell contains an entire organism’s genome. All cells of an organism have the same genome, but can have different phenotypes. For example, cells in your eye have the gene for producing hair protein (keratin) but this gene is not expressed in those cells. How do genes get switched on or switched off?

Gene regulation Where RNA polymerase binds (repressor protein prevents it binding – gene switched off) a) Gene switched off b) Gene active (as repressor protein can’t bind) Repressor protein can no longer bind

Why regulate gene expression? Cells conserve energy and materials by blocking unneeded gene expression. If a substrate is absent in the environment why produce the enzyme for that substrate! Repressor molecules keep the cell from wasting energy by not allowing transcription of mRNA to make proteins that have no use.

Homeotic genes Regulator genes that control gene expression during development Produce DNA-binding proteins that switch genes off, or some assist RNA polymerase to bind and therefore switch genes on. Mutations in these genes can result in extra fingers, limbs, missing body parts

Gene regulation in prokaryotes Bacteria have groups of genes that are controlled together and are turned on/off as required. E.g. the lac operon is a set of genes in bacteria used for lactose metabolism. Bacteria produce the enzymes to break down lactose into glucose and galactose but ONLY when lactose is present. http://www.sumanasinc.com/webcontent/animations/content/lacoperon.html http://pages.csam.montclair.edu/~smalley/LacOperon.mov

Lac Operon – an example of gene regulation in E. Coli The bacterium Escherichia coli is capable of producing the enzyme b-galactosidase which splits lactose to produce glucose and galactose. This enzyme is only produced when the bacteria encounters lactose. When lactose is not present, a protein binds to the promoter region of the b-galactosidase gene and prevents transcription (RNA polymerase cannot access the promoter). This protein is referred to as a repressor protein. When lactose is present in the growth medium of the bacteria, it enters the cell and binds to the repressor protein causing it to be removed from the DNA and allowing transcription to occur. The gene is ‘on’ or ‘off’ depending on the nutrients available to the cell.

Repressor protein lactose

Gene regulation in Eukaryotes Proteins produced by regulator genes Epigenetics Chemical modification: eg modification of histone proteins or methylation of DNA (switches genes on or off) RNAi (interference RNA) Binds to complementary sequences on mRNA Prevents that mRNA from being translated and the protein it codes for is no longer made

Epigenetics

Other factors in gene regulation Epigenetics shows that the environment of a cell can also influence the expression of genes. This includes factors such as: Light Temperature Ions Hormones Diet Medicines