Protein Synthesis (Transcription & Translation) and Mutations

What is the Central Dogma of molecular biology?

• DNA → mRNA → protein

• DNA carries the instructions to build all the proteins, but is transcribed into mRNA because it cannot leave the nucleus.

• In the cytosol, ribosomes translate the mRNA into a polypeptide chain.

What is gene expression?

Refers to the transfer of genetic information from DNA to RNA to protein.

What is RNA?

• AKA ribonucleic acid

• Nucleic acid

• Plays a role in creating protein

• Single stranded helix

What is the monomer for RNA?

Ribonucleotide

What is the structure of ribonucleotide?

• Ribose (pentose/ 5C sugar)

• Phosphate group

• Nitrogenous base

  • Adenine, guanine (purines, 2 rings)

  • Uracil, cytosine (pyrimidines 1, ring(

• Glycosidic bonds hold these components together

What are the 3 types of RNA?

• mRNA - Messenger

• tRNA - Transfer

• rRNA - Ribosomal

What are the 3 stages of protein synthesis?

Transcription, RNA processing (only in eukaryotes), and translation

What is mRNA?

• messenger RNA

• Single stranded

• Composed of ribonucleotide

• Carries the code for making proteins from DNA to the ribosomes in the cytoplasm

• The “blueprint”

What are the 3 parts of transcription?

Initiation, elongation, and termination

What is transcription?

• DNA → mRNA

• Occurs inside of the nucleus

• This process uses: DNA template strand, mRNA, RNA polymerase, mRNA

What is the template strand?

• Aka antisense strand

• The DNA strand being transcribed

What is the coding strand?

• Aka sense strand

• The DNA strand that is not being transcribed

What happens during initiation in transcription?

• RNA polymerase binds to the recognition site/promoter region of the DNA

• It unwinds the DNA and separates the template strand from the coding strand

What are the 3 key characteristics about the promoter region?

• Determines where transcription begins

• Usually rich in adenine and thymine (“TATA” box)

• Allows RNA polymerase to bind to the correct strand in the correct orientation

What happens in elongation in transcription?

• RNA polymerase encodes towards the 5’ end of the template strand

• It moves downstream, attaching ribonucleotides along the template strand following complementary base pairing

  • The coding strand and RNA are essentially the same sequence except….

  • A is paired with U rather than with T

• After transcription, pre-mRNA is formed, DNA immediately recoils, and RNA polymerase detaches

Note: Several RNA polymerases can work on a single gene at once

What happens in termination in transcription?

• RNA polymerase comes across the terminator sequence/signal

• The mRNA creates a “hair pin loop” which impairs RNA polymerase to keep going causing it to snap off

• Rho termination protein binds to the end of mRNA and moves toward RNA polymerase, eventually stopping it from transcribing

What happens in RNA processing?

• Only occurs in eukaryotes

• This stage protects the RNA from the caustic cytosol

• RNA gets a 5’ cap (or G cap) and 3’ poly-A tail

  • 5’ cap or G cap

    • Modified guanines added to 5’ end of mRNA (7-methyl guanosine)

  • 3’ poly-A tail

    • About 50-250 adenines are added to the 3’ end of mRNA

• Splicing additionally occurs

What happens in splicing?

• Spliceosomes contain proteins and snRNPs (small nuclear ribonucleic proteins)

• It rearranges exons (coding regions) and removes introns (non-coding regions)

• Alternative splicing allows the rearrangement of exons to produce different proteins when translated by ribosomes

• This process results in Mature mRNA

What are the 3 stages of translation?

Initiation, elongation, and termination

What happens in translation?

• mRNA → protein

• Occurs in the cytosol and/or often in the rough endoplasmic reticulum (RER)

• Ribosomes read the mRNA and then translate these instructions into an amino acid sequence

• This process uses: rRNA, tRNA, ribosomes,

What are ribosomes?

• Site of protein synthesis

• Composed of: 60% rRNA and 40% protein

• 2 subunits (small and large)

• E site = exit site - ejects

• P site = peptidyl site -pairs

• A site = acceptor/ aminoacyl site - attaches

What is a codon?

• 1 codon = a group of 3 consecutive bases on the mRNA transcript

• Codes for 1 amino acid

• Redundant: 2+ codons code for the same amino acid

  • 64 possible combinations of codons

  • But 20 amino acids, which make the code…

• Non-ambiguous: No codon codes for more than 1 amino acid

• Continuous: the code is read as a series of 3-letter codons

• Universal: Almost all organisms build proteins with the same genetic code

What is the start codon?

AUG (Methionine)

What are the 3 stop codons?

UAA, UAG, and UGA

What is tRNA?

• transfer RNA

• Attached to an amino acid by the enzyme aminoacyl-tRNA synthetase, forming an aminoacyl-tRNA complex

• Each tRNA molecule has an anticodon sequence, which is complementary to the mRNA transcript

What happens in initiation in translation?

• mRNA binds to the small ribosomal subunit (30S in prokaryotes or 40S in eukaryotes)

• The large ribosomal subunit (50S) binds to the complex

• The translation of the mRNA begins at 5’ end

• An initiator tRNA pairs with the start codon on mRNA

  • It is initially located on the P site

What happens in elongation in translation?

• A tRNA with the anticodon for the 2nd codon is placed at the A site

• The polypeptide chain is translocated from the tRNA at the P site to the tRNA on the A site

  • Resulting in the formation of a peptide bond by peptideyl transferase (A ribozyme)

  • This increases the growing polypeptide chain one amino acid at a time

• The ribosome shifts position one codon at a time and progresses toward the 3’ end of the mRNA

What is termination in translation?

• The process repeats until a stop codon (UGA, UAA, UAG) reaches the A site

• A release factor binds to the stop codon

• The polypeptide is hydrolyzed (released) from tRNA at the P site

• The entire complex disassembles

• mRNA can be reused

• The polypeptide is folded into a protein

What is a mutation?

Changes in the DNA sequence that are inherited

What are the 2 types of mutations?

• Gene mutations - those that produce change in a single gene

• Chromosomal mutations - those that produce changes in whole chromosomes

What are point mutations?

Involve changes in one or a few nucleotides and occurs at a single point on a gene

What are the 3 types of point mutations?

• Substitutions: One base is changed to a different base.

  • Usually affects one amino acid

  • Silent mutations - Substitution of the base has no effect because of:

    • The redundant nature of the genetic code

    • Substitution occurs in introns, which are cut out of the mRNA anyway, so it will never surface

  • Missense mutation

    • A change in the base sequence of DNA alters a codon

    • Leading to a different amino acid being placed in the protein sequence

    • e.g. sickle cell anemia

  • Nonsense mutation

    • A change in the DNA sequence causes a stop codon to replace a codon

    • Often lethal to the cell

• Frameshift mutation - a mutation that causes the reading frame of codons to change, usually resulting in different amino acids being incorporated into the polypeptide

  • Insertions: Point mutation in which one base is inserted into the DNA sequence

    • The effects can be dramatic

    • Codons are read in groups of 3 and adding one base makes the entire sequence to be misread

  • Deletions: Point mutation in which one base is deleted.

    • Deleting one base cause the entire sequence to be misread because they are still read in groups of 3

What are the 4 types of chromosomal mutations?

• Deletion: involves the loss of all or part of a chromosome.

• Duplication: produces an extra copy of all or parts of a chromosome

• Inversion: Reverse the direction of parts of a chromosome

• Translocation: Occurs when parts of one chromosome breaks off and attaches to another

What are the causes for mutations?

• Spontaneous mutations - occuring as a result of errors made in DNA replication

• Induced mutations - mutations caused by a chemical agent or radiation

  • mutagenic agents are chemicals or radiation which causes mutations