3.5 (SL)/ 7.3 (HL): Transcription

http://learn.genetics.utah.edu/content/begin/dna/firefly/

Compare the structure of DNA and RNA

Compare the processes of transcription and translation

DNA transcription (higher level)

Let’s begin with some animations…

1. from the Wellcome trust2. From PBS

Exceptions to the central dogma of genetics 1: retroviruses (thanks Max)

Prion protein replicationPrions are proteins that propagate themselves by making conformational changes in other molecules of the same type of protein

And finally….thanks, Ilona!

• exceptions

7.3.1: State that transcription is carried out in a 5’ – 3’ direction

Remember that BOTH transcription AND translation are carried out in a 5’ to 3’ direction

7.3.2: Distinguish between the sense and antisense strands of DNA

• Sense strand is the genetic code

• Sense strand is not copied

• Antisense strand is the template strand

• Antisense strand is used for transcription

• Antisense strand is complementary to sense strand

Sense and antisense DNA

• SENSE strand has same base sequence as transcribed mRNA, except that T is replaced by U

• RNA polymerase enzyme adds RNA nucleotides to sugar-phosphate background in 5’ – 3’ direction

Where does transcription begin?

• Promotor region: contains ‘promotor’ sequence: specific DNA recognised by activator proteins wh

Promotor Region

• Upstream of transcription site

• Contains specific DNA (TATAAA boxes) recognised by activation factors which recruit RNA polymerase

• 100 – 100 bases long• Very diverse in

eukaryotic DNA

Coding Region

• Provides specific triplet code for specific protein

• Coding region produces mRNA which will be modified after transcription

• mRNA produced by RNA polymerase

Terminator Region

• Site signals the end of DNA transcription

• Triplet code makes both RNA polymerase AND mRNA strand fall off the template

• Fold up mRNA strand to unlock both mRNA and polymerase

Process of transcription

• Animation of transcription• more complete animation of transcription

Post-translational modification• Eukaryotes only• mRNA is processed in the

nucleus before exportation to the cytoplasm

• Introns are removed (spliced)

• Mature mRNA is exported to the cytoplasm

• EXONS are joined together

Post-translational modification - splicing

• Animation on RNA splicing

Translation!

Let’s start with some animations…From John KyrkFrom WileyFrom Harvard

Welcome to the world of RNA!

Ribosomal RNA

Ribosomal RNA

Ribosomal structure form the PDB

Transfer (tRNA)• ‘Adaptor molecules’: one

end can read the triplet code on mRNA (anticodon), one end(3’) attaches to a specific amino acid (Crick, 1958)

• Each tRNA has a specific shape, determined by looping of helical sections

• Each tRNA specific shape fits one of 20 aa-tRNA synthase enzymes

Structure of tRNA• Amino acid attachment site

(3’) (acceptor loop/stem) :always CCA

• Complementary base pairing (H-bonds)

• Non-base pairing loops (7 or 8 bases)

• Anticodon (3 bases): atttaches to mRNA codon

• Mammals have 150 tRNA molecules

Activation of tRNA

• Activation of tRNA

How does the correct amino acid link to tRNA?

1. The shape of each tRNA is different, defined by folding of the loop and helical structures

RNA folding2. The shape of the tRA determines which of 20 specific

amino-acyl tRNA synthase enzymes it attracts3. ATP is needed to attach the amino acid to the 3’ CCA

end of the tRNA4. Each amino acid has >1 tRNA molecule (degenerate

code)

Structure of a ribosome (ribozymes)

• Ribosomes are actually ribozymes

• They catalyse translation of mRNA into a polypeptide

• The substrate is mRNA• Each ribosome is

multifunctional – it is not used up, and catalyse translation of many different mRNA codes

Structure of a ribosome

Prokaryotes and eukaryote ribosomes differ

Polysomes

Polysomes

• Cluster of ribosomes bound to mRNa strand

• Read mRNa simultaneously to increase efficiency of protein synthesis

• Direction of translation is 5’ to 3’

Free and fixed ribosomes

• Free ribosomes make proteins for ‘internal use’

• RER-bound ribosomes synthesise proteins which will be placed invesicles and exported from the cell

Translation has 4 stages

(leader sequence)

1.Initiation2.Elongation

3.Translocation4.Termination

(Leader sequence)

• Leader sequence contains a ribosome binding site

• In bacteria, called the Shine-Delgado box (AGGAGG)

• In vertebrates, the KOZAK box (170 bases)• Leader sequence contains many regulatory

sequences, including protein binding sites, that can affect stability of the mRNA or the efficiency of translation

Initiation

• mRNA has the start codon (AUG)

• Only one start codon• tRNA has anticodonUAC• Small ribosomal subunit associates with the tRNA• Large subunit moves

over mRNA

Initiation (2)

• ONLY AUG can occupy the P site ‘de novo’

• Next codon on A site if free to attract appropriate tRNA

Elongation

• Bond between tRNA and methionine is broken

• Energy released is used to form peptide bond

• GTP is also used as an energy source

• Large ribosomal subunit moves 3 base pairs (one codon) towards the 3’ end

Elongation (2)

• Both subunits are now over the P and A sites

• tRNA for methionine is on E site and is released for re-charging

• tRNA for proline is in P site

• A site is free and attracts tRNA for alanine

Elongation (3)

• Bond between tRNA and proline is broken

• GTP is provided as a source of energy

• Energy released forms peptide bond

• Ribosome moves along the mRNA towards 3’

Termination• Ribosome encounters s STOP

codon:• UGA, UAA, UAG• Not recognised by tRNA• Instead, release factors

(proteins) bind and facilitate release of the polypeptide from the translation process

• Two subunits move apart and separate

• Protein moves to ER and Golgi for processing