Teacher note

Nucleic Acids:

• DNA and RNA Nucleic acids are found in ALL living organisms.

• They are large polymers composed from nucleotide monomers which store genetic information and help produce proteins required for survival.

• There are TWO kinds of nucleic acid: • Deoxyribonucleic Acid (DNA) • Ribonucleic Acid (RNA)

DNA – the blueprint of life

• DNA is a very large molecule made up of a long chain of subunits called Nucleotides.

• Each Nucleotide is made up of:

  • A negatively charged phosphate group (PO4 )

  • A 5 carbon (pentose) sugar molecule – Deoxyribose

  • One of four nitrogenous bases

• There are four different kinds of nucleotides (nitrogenous bases) in DNA: • Adenine (A) • Thymine (T) • Cytosine (C) • Guanine (G) Cytosine (C) always pairs with Guanine (G) Adenine (A) always pairs with Thymine (T)

• Taking a closer look at the 5-carbon sugar: Each carbon is assigned a number in a clockwise direction (1’ – 5’)

• The three carbons of importance are: • 1’ (one prime) attaches to the nitrogenous base • 3’ (three prime) attaches to the phosphate of the following nucleotide • 5’ (five prime) attaches 5-carbon sugar to phosphate group of that nucleotide

• The bonds joining nucleotides are strong covalent bonds (phosphodiester bonds) which occur between the sugar group of one nucleotide and the phosphate group of the another.

• The two polynucleotide chains which make up the DNA molecule run in opposite directions – Antiparallel.

• During replication DNA Polymerase adds bases to the 3’ end. The molecule is assembled in the 5’ - 3’ direction.

• The two strands are twisted into a double helix.

• During replication DNA Polymerase adds bases to the 3’ end. The molecule is assembled in the 5’ - 3’ direction.

Histone and Nucelosome

• Histones are proteins found in Eukaryotic cells that tightly package DNA into structures called Nucleosomes.

• This ‘packaging’ causes the DNA double helix molecule to condense into a chromosome.

• Average length of DNA molecule in a human chromosome – 5cm.

• Average length of human chromosome 1 (fully condensed) – 5µm

• At the simplest level, chromatin is a double-stranded helical structure of DNA

• DNA is combined with histones to form a nucleosome

• Each nucleosome consists of eight histone proteins around which the DNA wraps 1.65 times

• The nucleosomes fold up to produce a 30nm fibre

• The fibre forms loops which are compressed and folded further

• This tight coiling of the fibre produces the chromatid of a chromosome

RNA – Ribonucleic Acid

• Consists of a single strand chain of unpaired nucleotides

• Includes four bases: • Adenine • Cytosine • Guanine • Uracil (replaces Thymine)

• RNA can serve many functions within a cell but is primarily involved in protein synthesis

Gene Structure and Expression

• Protein synthesis relies on the existence of the genetic code.

• The genetic code determines how genetic information (stored in genes) is transcribed and translated into functional proteins.

• By encoding sets of instructions on how to make various proteins, genes control the structure, biochemical and physiological functioning of an organism.

• The genetic code relies on the grouping of adjacent nucleotides into groups of 3.

  • In DNA – a group of 3 adjacent nucleotides is called a triplet.

  • When transcribed into mRNA – the 3 nucleotides are called a codon.

  • Each triplet or codon codes for a specific amino acid in the final polypeptide chain (protein molecule)

• To determine the amino acid coded for by a specific codon, a codon table can be used.

• A gene consists of a particular part of the DNA molecule

• However, only one of the two chains contains the information required to express the gene – Template Strand.

• The complementary strand is referred to as the coding strand.

Gene Structure

• A gene consists of several regions:

  • promoter region - upstream (5’ end) binding site for RNA polymerase

  • introns - non-coding regions of DNA

  • exons - coding segments of DNA

  • terminator sequence - signals end of transcription

  • operator – binding site for repressor proteins (inhibit protein synthesis)

Gene Expression

1. Transcription – copying of DNA into pre-mRNA

2. RNA processing – modifies pre-mRNA to produce mRNA

3. Translation -occurs on ribosomes in cytoplasm

   • involves decoding mRNA strand into polypeptide chain (protein)

Transcription (from DNA to pre-mRNA)

• Step 1: Initiation

  • RNA polymerase attaches to specific promoter sequence of template strand

  • DNA begins to unwind exposing bases of template strand

• Step 2: Elongation

  • RNA polymerase moves along DNA template strand

  • Complementary nucleotides are brought into place and joined one by one to form pre-mRNA chain (in the 5’ to 3’ direction)

• Step 3: Termination

  • When RNA polymerase reaches termination sequence, transcription stops

  • pre-mRNA is released from template

RNA processing

• Splicing: introns are ‘cut’ out and remaining exons are spliced together

• Capping: addition of methyl-guanine cap (methyl-G cap) at 5’ end. Cap protects mRNA from enzyme attack

• Adding a tail: chain of adenine nucleotides (poly-A tail) is added at 3’ end. Aids stability of mRNA

• Alternative splicing:

  • Single pre-mRNA strand can produce many different mRNA molecules depending on arrangement or removal of exons (allows for many different proteins to be created)

Translation

• mRNA moves from nucleus to cytoplasm where it attaches to ribosome.

• tRNA molecules bring amino acids to mRNA to be assembled into proteins.

• Transfer RNA:

  • tRNA consists of a single strand of 76 nucleotides coiled and paired

  • one end contains three bases (a triplet) which makes up an anti-codon

  • the other end contains a region that attaches to one specific amino acid

• Anti -Codon: Triplet unit located on tRNA

• Codon: Triplet unit located on mRNA

Trp Operon

• found in Escherichia coli (E coli)

• regulates the expression of structural genes which code for proteins involved in the production of tryptophan (amino acid)

• is an inducible operon (can be switched on or off depending on the levels of tryptophan present within the cell). This helps to conserve energy which is a finite resource

• The trp operon is composed of a series of structural genes (trpE, trpD, trpC, trpB and trpA) which are controlled by a common promoter and operator

• To regulate the expression of the structural genes, the regulatory gene for typ operon is constantly expressed

When tryptophan levels are high

• transcription of the trp structural genes is repressed in order to prevent unnecessary production of tryptophan

• When tryptophan levels are high, tryptophan binds to repressor protein causing conformational change which then allows repressor protein to bind to operator.

• Transcription of structural genes is inhibited therefore preventing production of tryptophan

When tryptophan levels are low

• transcription of the trp structural genes is activated in order to increase amount of tryptophan available

• When tryptophan levels are low, there is an insufficient quantity of tryptophan available to bind to repressor protein.

• Repressor protein detaches from operator allowing transcription of structural genes to occur.

• Tryptophan levels rise.

Attenuation

• Attenuation is a second mechanism used to control expression of the trp operon

• mRNA transcribed from the trp operon has a leader sequence upstream of the coding region of the trpE structural gene

• This leader sequence encodes a 14-amino acid leader peptide containing two trp molecules

• The leader sequence contains four regions/domains (numbered 1–4) that can form one of two base paired stem-loop (‘hairpin’) secondary structures

• If pairing occurs between regions 3 and 4, then an attenuator is formed and transcription is terminated

• If pairing occurs between regions 2 and 3, then the attenuator does NOT form and transcription continues

When tryptophan is abundant

• When tryptophan is plentiful, translation does not stop at the 2 trp codons, it continues until it reaches a stop codon that is located between regions 1 and 2

• The position of the stop codon prevents region 2 from pairing with region 3

• Region 3 therefore pairs with region 4 and an attenuator is formed- only the leader peptide is translated

• The remainder of the structural genes are not transcribed or translated – tryptophan is not produced

When tryptophan is scarce

• When tryptophan is in short supply, the ribosome pauses at the two trp codons located in region 1

• This leaves region 2 free to pair with region 3 (to form an anti-terminator)

• If regions 2 and 3 are paired up, region 3 cannot pair with region 4

• Transcription continues to the end of the trp operon – tryptophan is produced