Genetics - DNA replication, transcription and translation

In which cellular locations does protein synthesis occur?

• Eukaryotic cells - nucleus and cytoplasm

• Prokaryotic cells - cytosol

What was the Pulse-Chase Experiment and what did it lead to?

• Paul Zamecnik and Sydney Brenner were investigating how proteins were made in cells in the 1960s

• Cells were exposed to radioactive uracil

• RNA was synthesised with this uracil

• RNA moved from the nucleus to the cytoplasm where proteins are synthesised

• First clue that RNA was a messenger

• RNA transports genetic instructions out of the nucleus to the ribosomes

• Would later become known as messenger RNA (mRNA)

• Genetic instructions carried by DNA must be transcribed into RNA

What was Ochoa and Kornberg’s work on RNA/DNA synthesis?

• Discovered the enzymes responsible for synthesizing RNA and DNA (RNA and DNA polymerase)

• Earned the Nobel Prize in 1959

How is RNA different to DNA in its chemical structures?

• DNA uses deoxyribose sugar

• RNA uses ribose sugar

• Uracil has the same base-pairing properties as thymine but is present in RNA only

• RNA is more chemically reactive than DNA

• Ribose sugar has an OH on its 2’ carbon where deoxyribose has an H

• DNA is used for accurate, long-term storage

• RNA is used for transient activities before it is broken down

How is RNA different to DNA in its overall structure?

• RNA is mainly single-stranded

• Contains small sections of paired strands between complimentary base pairs

• RNA can form a variety of 3D shapes (hairpins and loop structures)

• RNA can base pair with other nucleic acids through non-conventional base pairing

How is RNA different than DNA in its function?

• Many types of RNA with many functions, including catalysis

• Messenger RNA (mRNA) codes for proteins

• Ribosomal RNA (rRNA) form the core of ribosomal structure and catalyses protein synthesis

• MicroRNA (miRNA) regulates gene expression

• Transfer RNA acts as an adaptor between mRNA and amino acids during protein synthesis

• Noncoding RNA is used in RNA splicing, gene regulation, telomere maintenance and more

What is the process of transcription?

• RNA is synthesised from a DNA template by RNA polymerase (formed from ribonucleoside triphosphates)

• RNA Polymerase I transcribes rRNA

• RNA Polymerase II transcribes mRNA - process of transcription

• RNA Polymerase III transcribes tRNA

• RNA is synthesised in a 5’ to 3’ direction (antiparallel to its template DNA strand)

In which type of cell is mRNA processed from pre-mRNA to mature RNA?

Eukaryotic cells

• Bacterial DNA does not contain introns so does not need processing before translation

What is the process of mRNA processing?

• Processed before it leaves the nucleus

• Processing of the ends of mRNA is essential for stability

• 5’ end is capped with an atypical nucleotide

• 3’ end is capped with a poly-A tail

• Introns are removed from the RNA strand

• Processes take place as the mRNA is transcribed

How are introns removed from mRNA?

• Introns are removed by the spliceosome

• Splicing reaction is performed by small nuclear RNA molecules (snRNA)

• Bind to proteins to form small nuclear ribonuclearprotein particles (snRNPs)

• snRNPs form the core of the spliceosome and assist in directing splicing

What is alternative splicing and how is it beneficial?

• Alternative splicing produces two separate isoforms from the same pre-mRNA molecule

• Allows more than one protein to be expressed from a single gene

• Transcripts of ~95% of genes exhibit alternative splicing

• Occurs when the same gene is expressed in different cell types

When is mRNA exported from the nucleus?

• Mature mRNA is selectively exported from the nucleus

• RNA binding proteins mark a mature and intact mRNA for export from the nucleus

• Only a small fraction of synthesised mRNA is exported

• Incorrectly synthesised mRNA molecules are broken and the nucleotides are reused

How does mRNA stability affect translation rates?

• mRNA can be translated many times

• Stability determines how much protein is translated in the cell

• Sequence of mRNA affects its half life

  • Some have lifetimes of over 10 hours

  • Some have lifetimes of less than 30 minutes

How did the COVID pandemic help geneticists understand the genetic code better?

• Scientists needed a quick solution

• Traditional vaccines take years to develop

• mRNA vaccines were developed in record time using the principles of the genetic code

How were mRNA vaccines against COVID developed?

• SARS-CoV-2 virus carries genetic information in the form of RNA

• Scientists sequenced this viral RNA to identify the part of the code that codes for the spike protein which allows human cell infection

• Used the universal genetic code to create synthetic mRNA

• mRNA would serve as a ‘blueprint’ for the spike protein but not the active virus

• Spike proteins were enough to teach the immune system what to recognise and attack

• When the mRNA is injected into the body, it enters cells and undergoes translation

• Ribosomes in the cells read the mRNA using the genetic code to produce the viral spike protein

• Protein isn’t harmful on its own, immune system detects it and learns how to defend against the real virus

What are the benefits of producing an mRNA vaccine?

• Don’t require the growth of live viruses

• Make them faster and safer to produce

Who were scientists that aided in discoveries surrounding the process of translation?

• Har Gobind Khorana

• Indian-American biochemist

• Shared the 1968 Nobel Prize for Physiology or Medicine with Marshall W. Nirenberg and Robert W. Holley for research that showed the order of nucleotides in nucleic acids

• Leslie Barnett, Francis Crick, Sydney Brenner and Richard Tobin

• Discovered elements relating to the triplet code in translation

What did Crick, Brenner, et al.’s experiment involve?

• Infected E. coli with Bacteriophase T4

• Mutations in the rII region in the bacteriophage cause changes to the plaques formed

• Generated mutants in the rII gene using proflavin (planar molecule that intercalates between base pairs and can cause a single indel mutation)

• Brought mutants of opposite types together by recombination to produce a mix of single and recombinant mutants

• A single ± mutation caused mutants due to frameshift

• A double ± mutation did not cause mutants

• Double mutations of the same type caused mutants

• Recombining 3 mutants of the same type result in a wild-type (non-mutant) as frameshift is reverted back to normal reading

What are the main properties of the genetic code?

• Degenerate - all possible combinations are used to code for an amino acid (some will code for more than one amino acid)

• Non-overlapping - single base mutations only ever affect one amino acid

• Universal

What is the reading frame of RNA?

• Read in sets of three nucleotides (codons)

• Genetic code was deciphered in biochemical experiments by Marshall Nirenberg and Har Gobind Khorana

• tRNA were described by Robert Holley

• All three shared a Nobel Prize in 1968

• Some amino acids have more than one tRNA associated with them

• Three codons do not code for amino acids but are instead stop codons

• Single letter code for amino acids allows protein sequences to be analysed digitally (bioinformatics)

What are the three stages of translation?

• Initiation

• Elongation

• Termination

What are the roles of RNA molecules in translation?

• mRNA carries the genetic information

• tRNA deciphers the mRNA codons

• rRNA makes up the ribosome

What is the structure of tRNA and its function?

• Clover leaf structure of single-stranded RNA

• Some tRNA molecules can tolerate a mismatch at the third codon position (‘wobble’)

• Genetic code is translated by aminoacyl-tRNA synthetases and tRNAs

• Each synthetase couples a particular amino acid to its corresponding tRNA (charging)

What is the function of rRNA?

• Associates with a set of proteins to form the translational machinery

• Ribosome is a large complex of four RNA molecules and more than 80 proteins

• RNA molecules in the structure choreograph and catalyse protein synthesis

• 3 sites within the ribosome

• A-site — aminoacyl-tRNA site

• P-site — peptidyl-tRNA site

• E-site — exit site

What happens during the initiation phase of translation?

• Translation initiation factors and initiator tRNA bind to the small ribosomal subunit with translation initiation factors bound

• mRNA binds to the small subunit

• Small ribosomal subunit with bound initiator tRNA moves along mRNA looking for the first AUG codon (Met amino acid)

• Translation initiation factors dissociate

• Charged tRNA binds to a codon site

• First peptide bond forms

What happens during the elongation phase of translation?

• tRNA molecules bind to the P-site and A-site

• Peptide bond forms between the two amino acids (amino to carbonyl groups)

• Large subunit translocates down the mRNA (E-site becomes P-site, P-site becomes A-site)

• tRNA is ejected once unbonded from its amino acid

What happens during the termination phase of translation?

• Release factor binds to the A-site

• Water binds to the C-terminus of the last amino acid

• Ribosome dissociates from the mRNA strand

In which direction does translation occur?

N to C direction (5’ to 3’)