Protein Synthesis

• Gene expression → process by which DNA directs the synthesis of proteins and synthesis of RNA.
  • Includes two stages: transcription and translation.

Stage 1: Transcription:

• Genes provide instructions to make a protein, but cannot build a protein directly.
• RNA → single stranded, is the bridge between DNA and protein synthesis.
  • Contains ribose instead of deoxyribose as its sugar & has uracil base instead of thymine.
• Sequences of nucleotides (aka the monomers of nucleic acids) convey information.
• Transcription → the synthesis of RNA using information from DNA.
  • Information from DNA is “rewritten” to RNA by serving as a template for a complementary sequence.
• The resulting RNA molecule (known as mRNA) serves as a transcript of the gene’s protein building instructions; serves as a “messenger”, hence the name “messenger RNA (mRNA)”

A Closer Look at Transcription

• mRNA is transcribed via the enzyme RNA polymerase (or RNA polymerase II).
  • This pries the two DNA strands apart and joins RNA nucleotides that are complementary to the DNA strand.
• It can only assemble a polynucleotide in the 5’ → 3’ direction.
  • Can assemble it from scratch, does not require a primer.
• Has three stages: initiation, elongation, & termination.
• Initiation → It starts at the promoter sequence (which is usually TATA), that includes the transcription start point where the RNA polymerase will bind to and start.
  • Transcription factors are used in eukaryotes where they help the RNA polymerase bind to the promoter. They bind first, and then the RNA polymerase binds. This whole arrangement is known as the transcription initiation complex.
• Elongation → As RNA polymerase moves throughout the DNA, it will unwind the double strands, which exposes 10-20 DNA nucleotides at a time to pair with RNA. RNA polymerase will then build RNA nucleotides towards the 3’ end.
  • The newly synthesized RNA will then peel away from the DNA template and the DNA double helix will reform.
  • Done at a rate of 40 nucleotides per seconds (eukaryotes).
• Termination → This is done differently in bacteria and eukaryotes.
  • Bacteria → proceeds through a termination sequence in the DNA. The transcribed terminator will act as a signal for the RNA polymerase to detach from the DNA and release the mRNA.
  • Eukaryotes → RNA polymerase transcribes the polyadenylation signal sequence (AAUAAA) in the pre mRNA. Proteins will then bind to the sequence where it will cut the RNA transcript free 10-35 nucleotides down from the signal. The RNA polymerase will continue to transcribe until enzymes catch up to dissociate the polymerase.

Stage 1.5: RNA Processing (In Eukaryotes):

• Enzymes modify the pre-mRNA before it leaves the nucleus, such as altering the ends and splicing certain interior sections.

Alteration of mRNA Ends:

• The 5’ end of the RNA is given a 5’ cap, which is a modified form of a guanine nucleotide added on the 5’ end.
• An enzyme adds 50-250 adenine nucleotides to the 3’ end, which forms a poly-A tail.
• Both of these facilitate the export of the mRNA out of the nucleus, prevent it from degradation by hydrolytic enzymes, and help ribosomes attach the 5’ end of the RNA.
• These will not be translated into proteins, along with several untranslated regions (UTRs)

Split Genes & RNA Splicing:

• RNA splicing → removal of large portions of RNA transcript and gluing the remaining portions together.
  • Think of it like editing a movie before it gets released.
• Primary RNA transcripts are 27,000 nucleotides long, but average proteins only require 1,200 RNA nucleotides to code for ~400 amino acids.
• RNA transcripts have long stretches of nucleotides that are uncoded and end up not being translated, these are called introns.
  • Introns are found in between coding segments that have expressed sequences, known as exons.
• Spliceosome → a complex made from small nuclear ribonucleoproteins (snRNPs) that removes introns by binding to nucleotide sequences and joins exons together.
  • The snRNPs participate in the splicing, recognize splice sites at introns, and catalyze the splicing reaction.

Stage 2: Translation:

• Translation → synthesis of a polypeptide using mRNA, done in the ribosome.
• Triplets of nucleotides are used to code for amino acids, known as codons.
• Codons are used to be translated into a sequence of amino acids that will make up a polypeptide.
  • Read in the 5' → 3’ direction.
• A codon chart is used to determine the 20 amino acids that the codon could potentially code for.

A Closer Look at Translation:

• In translation, a cell reads the series of codons in mRNA and uses tRNA to translate it.
  • tRNA transfers amino acids from the cytoplasmic pool to a growing polypeptide chain.
• tRNA has a specific amino acid at one end of its structure and a complementary nucleotide to the mRNA on the other side. This makes it so that the tRNA can translate mRNA codons to amino acids.
  • Consists of a single 80 nucleotide long RNA strand that can fold on itself to form a 3D structure.
  • Contains an anticodon → a nucleotide triplet that is complementary to an mRNA codon.
• mRNA is translated as tRNAs position each amino acid in the order described and the ribosome adds that amino acid on the growing polypeptide chain.
  • The matching up of tRNAs and amino acids is done by aminoacyl-tRNA-synthetases, an enzyme where they join an amino acid to the right tRNA through covalent attachment via hydrolysis of ATP. This results in a charged tRNA, which is released from the enzyme and delivers the amino acid to the growing polypeptide.
  • Base pairing between the third base of an mRNA codon and the corresponding tRNA anticodon is more relaxed, which is why some codons code for the same amino acid.
• Ribosomes facilitate the coupling of anticodons and codons. They consist of a large subunit and a small subunit, which is both made of ribosomal RNA (rRNA).
  • The ribosome has special binding sites for translation.
    • The P-site (peptidyl-tRNA) holds the tRNA with the growing polypeptide.
    • The A-site (aminoacyl-tRNA) holds the tRNA with the next amino acid that will be added to the polypeptide chain.
    • The E-site (exit) is where discharged tRNA exits the ribosome.
• Translation is done in 3 stages: initiation, elongation, and termination. Initiation and elongation require energy in the form of guanosine triphosphate (GTP)
  • Initiation → a ribosomal subunit brings the mRNA and the tRNA w/ the first amino acid together. The subunit starts at the 5’ end of the mRNA and “scans” until it reaches the AUG start codon, where the tRNA will bind to. AUG signals the start of translation. A large ribosomal subunit is then binded using GTP, creating the translation initiation complex.
  • Elongation → amino acids are added via elongation factors and are moved in the 5’ → 3’ direction.
  • Termination → Elongation continues until it reaches the UAG, UAA, or UGA codons, which signal the end of protein synthesis. A release factor then binds to the stop codon and adds a water molecule, which breaks the bond between the completed polypeptide chain and the tRNA.

Structure of Proteins

• Protein → a biologically functional molecule made up of one or more polypeptide chains.
• Proteins are constructed from the same 20 amino acids, which are bonded by peptide bonds.
  • A polymer of amino acids is called a polypeptide chain.
• The function and activities of proteins are determined by their structural shape by which the polypeptide chains are twisted, folded, and coiled into a specific shape.
  • The function of a protein depends on its ability to recognize and bind to some molecule.
    • For example, antibody proteins can bind to proteins from viruses.
• Polypeptide chains may fold spontaneously during synthesis, which is driven by the amino acids (in other words, amino acid sequence determines protein shape).
  • The protein environment can also influence the structure. Changes in pH, salt concentration, or temperature can impact the stability and support of the structure. Proteins that lose their shape are called denatured.
  • Misfolding of the protein can happen; this leads to diseases such as cystic fibrosis, Alzhiemer’s, Parkinson’s, and mad cow disease.
• Many proteins are roughly spherical (globular), while some are shaped like long fibers (fibrous)

Four Levels of Protein Structure

• Proteins have usually 3 levels of structure: primary, secondary, and tertiary. Proteins consisting of 2 or more polypeptide chains have a quaternary level.
• Primary structure → This is the linear sequence of amino acids. For example, transthyretin, a blood protein, would have a sequence of 127 amino acids.
  • It’s important to note that the primary structure is not determined by the linking of amino acids, but by inherited genetic information via mRNA.
  • Dictates secondary & tertiary structure.
• Secondary structure → These are regions that are stabilized by hydrogen bonds between atoms in the polypeptide backbone. These include segments of the polypeptide chains that are repeated coils and folds. The repeated hydrogen bonds create these as the oxygen atoms have a partial negative charge and the hydrogen atoms attached to nitrogens have a partial positive charge in the backbone.
  • 𝛼 helix → a coil that is held together by hydrogen bonds between every 4th amino acids. Some proteins have multiple of these, such as 𝛼-keratin, which has 𝛼 helix formation over most of its length.
  • ꞵ pleated sheet → two or more segments of the polypeptide chain lying parallel to each other connected by hydrogen bonds. They make up the core of many globular proteins.
• Tertiary structure → this is the overall 3D shape of the polypeptide, which results from interactions between the R groups/side chains of various amino acids.
  • Hydrophobic interactions contribute to the tertiary structure by the exclusion of nonpolar substances by water. As a polypeptide folds into its shape, amino acids with hydrophobic side chains converge at the core so that they stay away from water. They stick together via Van der Waals interactions.
  • Disulfide bridges reinforce the shape of the protein by forming where 2 cysteine monomers are brought together by folding. The sulfur of one cysteine attaches to the other sulfide, and the disulfide bridge acts as a rivet to hold them together.
• Quaternary structure → this is the overall aggregation of multiple polypeptide chains. For example, transthyretin is made up of 4 polypeptide chains.
  • Hemoglobin also has quaternary structure, with 4 polypeptide subunits, two which are 𝛼 and two that are ꞵ.

Enzymes

• Enzyme → a macromolecule that acts as a catalyst, or a chemical agent that speeds up a reaction without being consumed.
• Enzymes end with “-ase”, such as polymerase or sucrase.
• Enzymes lower the activation energy needed to start a reaction.
• Are specific to the reactions they catalyze.
  • For example, sucrase only works on sucrose and will not bind to any other disaccharides.
• Substrate → The reactant that the enzyme works on. The enzyme binds to the substrate, which forms an enzyme substrate complex.
  • When the enzyme & substrate join together, the enzyme converts the substrate to the products of that reaction and then releases those products.
• The enzyme binds to the substrate at a specific region called the active site, which is typically a pocket or groove where the substrate fits and catalysis occurs.
  • The enzyme will change slightly to make the substrate fit more; this is called induced fit.
• The rate at which the enzyme converts substrate to product depends on the initial concentration of substrate.
  • The more substrate molecules, the more frequently the conversion to product will occur.
  • However, at one point, the substrate concentration will be high enough so that all the active sites are open, which will cause the enzyme to be saturated.
• Temperature and pH levels also affect enzyme activity. The rate of an enzymatic reaction increases as temperature increases, but once the temperature reaches a certain point (or the optimal temperature), the rate will decrease. The optimal pH values for high enzyme activity is 6-8.
• Enzymes require non protein molecules for help for catalysis activity. These are called cofactors and can either be bound permanently to the enzyme or may bind loosely to it.
  • Cofactors can be inorganic, such as zinc, iron, or copper, but can also be organic, in which they are called coenzymes.

Mutations

• Mutation → a change in the nucleotide sequence in the DNA.
• Mutations can lead to changes in protein structure and function and can be passed down to offspring.
  • Mutations that impact the phenotype are called genetic disorders. For example, sickle cell disease is a genetic disorder that is caused by a mutation.

Small Scale Mutations:

• Point mutations → a change in a single nucleotide of a gene.
• Nucleotide-pair substitution → when one nucleotide gets replaced by another.
  • This can code into a missense or nonsense mutation. Nonsense mutation is when the new nucleotide creates a codon that codes for a STOP. A missense mutation is when it leads to a new amino acid being coded. There are 3 types of missense mutations:
    • Silent → when the same amino acid gets coded.
    • Conservative → when a different amino acid, but from the same type gets coded.
    • Non conservative → when a different amino acid gets coded.
• Frameshift mutation → when an insertion or deletion of a nucleotide ends up changing the arrangement of codons.

Large Scale Mutations:

• Translocation → when 1 gene on 1 chromosome is swapped for another chromosome from a different gene.
• Inversion → where two genes on a chromosome swap places. Breaks off and flips orientation.
• Monosomy → absence of genes in a chromosome.
• Trisomy → Addition of half a chromosome.
• Deletion → part of a chromosome is removed.
• Duplication → part of a chromosome is present in 2 copies.

Mutagens & Carcinogens:

• Mutagen → a chemical substance or physical event that can cause genetic mutations.
  • Endogenous → found inside the body
  • Exogenous → found outside the body and in the external environment.
• Carcinogen → a chemical substance that causes cancer by causing mutations in the cell’s DNA to reproduce faster or by increasing the rate of growth in a cell.