Protein Synthesis: Transcription and Translation

• Lecture 9 focuses on the sequence of events in protein synthesis.
• This includes transcription, pre-mRNA processing, the DNA sequence to protein sequence link, and translation.

Gene Expression

• Gene expression involves the process of converting DNA into a functional product.
• Genotype refers to an organism's hereditary information.
• Phenotype refers to actual observable or physiological traits.
• Phenotype is determined by the interaction of genotype and environment.
• DNA stores and transmits heritable information from generation to generation.
• RNA acts as a messenger, allowing the information stored in DNA to be used to make proteins.
• Proteins perform cellular functions.
• The three main steps of gene expression are transcription, processing, and translation.

Transcription: Overview

• Transcription involves the creation of RNA from DNA.
• Initiation begins with polymerase binding to a promoter.
• Elongation involves the polymerase moving downstream.
• Termination involves the release of RNA.
• RNA uses Uracil instead of Thymine.
• RNA is single stranded, while DNA is double stranded.

Transcription: Initiation

• Assembly of multiple proteins is required before transcription can commence.
• A eukaryotic promoter includes the TATA box, typically 25 nucleotides upstream.
• Transcription factors bind to DNA, including the TATA box binding protein (TBP).
• RNA Polymerase II binds along with more transcription factors, forming the transcription initiation complex.

Transcription: Elongation & Termination

• 10-20 nucleotides are exposed at a time, as a DNA unwinds.
• RNA nucleotides are added to the 3' end of the growing transcript forming a phosphodiester bond.
• The DNA double helix reforms as the transcript leaves the template strand.
• Transcription ends after transcription of the polyadenylation signal (AAUAAA).
• After transcription, the pre-mRNA transcript is ready for further processing.

mRNA Processing

• mRNA processing involves capping, tailing, and splicing.
• Capping involves adding a modified guanine nucleotide to the 5' end.
• Tailing involves adding 50-250 adenine nucleotides (poly-A) to the 3' end.
• Capping and tailing facilitate export, stability and ribosome binding in the cytoplasm.
• Splicing involves removing introns from the transcript.
• Exons are regions that remain in mature RNA, with UTRs (untranslated regions) at the 5' and 3' ends.
• Introns are intervening regions that do not remain in mature RNA.
• Splicing occurs in the spliceosome within the nucleus.
• Alternative splicing allows for multiple gene products from the same gene.

Gene and Product Size

• Genes and their products can vary in size.
• TATA-box binding protein (TBP) spans 18,000 base pairs on chromosome 6 and has 8 exons.
• Huntingtin (HTT) spans 180,000 base pairs of chromosome 4 and has 67 exons.
• The human genome contains ~3000 million base pairs and ~20,000 genes.

Sequence

• Protein sequence determines its final structure, which then determines its function.
• DNA mutations can affect the protein's ability to function.

Translation: Overview

• Mature mRNA transcript exits the nucleus and is bound by the ribosome for translation.
• Codons are translated into amino acids.
• tRNA molecules carry corresponding amino acids.
• Hydrogen bonds form between mRNA and the tRNA anticodon.
• Amino acids are added via peptide bonds.
• Translation includes the steps of initiation, elongation, and termination.

Translation: Ribosome Binding

• tRNA and mRNA are held within the ribosome to enable polypeptide formation.
• mRNA binds to a small ribosomal subunit.
• The A site holds the "next-in-line" tRNA.
• The P site holds the tRNA carrying the growing polypeptide.
• The E site is where tRNAs exit.

Translation: tRNA

• tRNA is the physical link between the mRNA and the amino acid sequence of proteins.

Translation: Initiation

• The initiator tRNA carries methionine (Met).
• A small ribosomal subunit with the initiator tRNA binds the 5' cap of mRNA.
• The small ribosomal subunit scans downstream to find the translation start site (AUG).
• Hydrogen bonds form between the initiator anticodon and mRNA.
• Completing the initiation complex require a large ribosomal subunit binds.

Translation: Elongation

• Codon recognition occurs through base pairing with a complementary anticodon.
• GTP is invested to increase accuracy.
• An rRNA catalyzes peptide bond formation.
• Translocation occurs, in which tRNA moves from the A site to the P site.
• tRNA in the P site moves to the E site and is released and requires energy (GTP).
• Empty tRNAs are reloaded in the cytoplasm using aminoacyl-tRNA synthetases.

Translation: Termination

• The process begins when the ribosome reaches a stop codon on the mRNA.
• A release factor promotes hydrolysis and releases the polypeptide.
• Ribosomal subunits and other components dissociate, and hydrolysis of two GTP molecules is required.

Gene Expression Regulation

• Gene expression can be regulated at multiple control points.
• These points include assembly of transcription factors, accessibility of DNA, capping, extent of polyadenylation, alternate splicing, nuclear export of mRNA, regulatory proteins, variable mRNA life-spans.

Gene Expression Control

• Control of gene expression is important to achieve the right action, at the right time, in the right place.
• Housekeeping proteins are continuously produced in large quantities and have longer half-lives.
• Other proteins are produced in response to stimuli.

Amino Acid Properties

• Amino acid side chains (R groups) determine the properties of each amino acid.
• Twenty standard (coded for) amino acids are classified by their side chain properties.
• Amino acid properties collectively determine the final structure and function of the protein.

Protein Primary Structure

• Primary structure is the sequence of amino acids.
• Polypeptide starts to form secondary structures as soon as it leaves the ribosome.
• Peptide bonds are covalent bonds between amino acids.
• Protein sequence (primary structure) is determined by DNA sequence.
• DNA and RNA are read from 5' to 3'.

Protein Structure

• Secondary structure includes alpha helices and beta pleated sheets, held by weak hydrogen bonds.
• Tertiary structure is the 3D shape stabilized by hydrogen bonds and side chain interactions.
• Quaternary structure refers to multiple proteins associating together to form a functional protein.

Protein Processing and Sorting

• Translation commences on free ribosomes.
• Many proteins are processed and sorted through the rough ER (RER) and Golgi.
• Proteins destined to function in the cytosol complete translation on free ribosomes.
• Proteins that go through the endomembrane system complete translation at fixed ribosomes on the RER.

Ribosomes to RER

• Signal peptides direct ribosomes to the RER.
• Signal peptide is located at the N terminus of the protein (~20 amino acids).
• A secretory protein is soluble in the lumen, while a membrane protein remains anchored to the membrane.

Post-Translational Modifications

• Translation is complete, but proteins may not be functional without post-translational modifications.
• Common post-translational modifications include phosphorylation, methylation, acetylation, biotinylation, carboxylation, carbohydrate addition, cleavage, and ubiquitination.
• Modifications can confer activity, the ability to interact, or direct proteins to locations, and some error cause issues.