Gene Expression and Protein Synthesis

Gene Expression and Protein Synthesis

• The most controlled and best understood part of gene regulation is transcription.

DNA to Protein Synthesis

• The DNA molecule stores information, which is analogous to a cookbook.
• The genes are the pages in the cookbook.
• Recipes used to prepare a meal are like the genes needed to provide an inheritable trait.
• The information stored in DNA is expressed through the combination of amino acids for protein synthesis.
• The central dogma of molecular biology states that DNA information is transferred to RNA, then expressed in the structure of proteins.
• Gene expression involves transcription and translation.
• Transcription is when the information is transfered to create a complementary strand of RNA.
• Translation is the process in which the mRNA nucleotide sequence is used to assemble a specific protein

Gene Expression Variations in Viruses

• Some viruses' gene expression deviates from the central dogma.
• RNA viruses replicate RNA from RNA, while retroviruses reverse-transcribe RNA into DNA.

Transcription Basics

• The DNA information must be carried out of the nucleus
• Transcription is analogous to copying a recipe
• Transcription accomplishes this task by transcribing the information from DNA into RNA, specifically messenger RNA (mRNA).
• Messenger RNA carries the genetic instructions from the nucleus to the site of protein synthesis.
• Ribosomal RNA (rRNA) is needed to form ribosomes.
• Transfer RNA (tRNA) is essential for translating nucleotide language into protein language.

Translation Overview

• Messenger RNA acts as a template for amino acid assembly.
• The nucleotide language is translated into the amino acid language with high accuracy.
• Transfer RNA facilitates translation.
• Each three-base sequence in RNA corresponds to an amino acid; this is the genetic code.
• Transcription and translation occur sequentially in eukaryotes, transcription in the nucleus and translation in the cytoplasm.
• Prokaryotes lack a nucleus, so transcription and translation occur simultaneously in the cytoplasm.
• Simultaneous transcription and translation can occur.
• Eukaryotic gene expression and protein synthesis are focused on because of their relevance to human health.

Transcription of DNA

• Transcription begins when DNA unwinds near the gene needing transcription, initiated by the enzyme helicase.
• RNA formation uses only one DNA strand, called the template strand, (-) strand, or antisense strand.
• The other strand is called the coding strand, (+) strand, and sense strand, matches the RNA sequence.
• Coding strand and template strand are the most commonly used for referring to the + and - strands respectively.
• Ribonucleotides assemble along the unwound DNA strand in a complementary sequence
• The + and - strands are complementary; where C on DNA pairs with G on mRNA, G pairs with C, A pairs with U, and T pairs with A.
• Eukaryotes use three RNA polymerase types for transcription.
• RNA polymerase I (pol I) mostly forms rRNA.
• Pol II catalyzes mRNA formation.
• Pol III forms tRNA and some regulatory RNAs like snRNA.
• Each polymerase is a complex of 10+ subunits, some unique, others shared.
• Eukaryotic genes include structural genes (transcribed into RNA) and regulatory portions (control transcription).
• Genes include exons and introns.
• Introns are removed, exons remain.

Transcription Control Elements

• Transcription includes the promoter, which has a unique base sequence on the DNA strand recognized by polymerase as an initiation signal.
• Besides unique nucleotide sequences, promoters contain consensus sequences like the TATA box.
• The TATA box, with the sequence beginning TATAAT, lies about 26 base pairs upstream from transcription start.
• Sequences of DNA are conventionally given from the coding strand for transcription description.
• TATA boxes are common to all eukaryotes.
• RNA polymerases interact with promoter regions through transcription factors (binding proteins).
• Enhancers speed up transcription, binding to DNA sequences distant from the transcription site and binding to transcription factors.
• Elongation is the process where RNA polymerase joins complementary bases, forming a phosphate ester bond between each ribose and the next phosphate group.
• Termination sequences signal the end of transcription.
• The enzyme Pol II terminates transcription.
• Pol II exists in phosphorylated (elongation) and unphosphorylated (initiation) forms, recycled via phosphorylation and dephosphorylation.
• mRNA synthesis occurs from the 5' to 3' end; complementary RNA and DNA run oppositely
• the enzyme moves along the DNA template strand in the 3' → 5' direction.
• As RNA synthesizes, it separates from the DNA template and the double helix reforms.
• Transfer and Ribosomal RNA are also similarly synthesized

RNA Processing

• RNA transcription products are not necessarily functional RNAs like mRNA but contain exons and introns.
• Functional mRNA requires transcript modification at both ends.
• The 5' end gains a methylated guanine (7-mG cap).
• The 3' end has a poly-A tail of 100-200 adenine residues.
• Introns are removed via a post-transcription process after capping.
• Transcribed tRNA is trimmed, modified, and methylated to become functional.
• Functional rRNA undergoes post-transcriptional methylation.

Translation of RNA

• Translation converts the mRNA genetic code into the amino acid sequence of proteins.
• mRNA, rRNA, and tRNA participate in translation, which occurs on ribosomes which dissociate into larger and smaller bodies.
• The spheres dissociate into two parts—a larger and a smaller body.
• Each of these bodies contains rRNA and some polypeptide chains that act as enzymes, speeding up the synthesis.
• In higher organisms, including humans, ribosome subunits called 60S and 40S; prokaryotes have 50S and 30S subunits.
• Messenger RNA binds to the smaller ribosomal body, is later joined by the larger body and stretches out.
• Codons are triplets of bases on mRNA where each amino acid is carried by a specific tRNA molecule.

tRNA structure

• Transfer RNA molecules contain a site to which enzymes can bind amino acids and also a recognition site for recognition
• The 3' terminus of tRNA is single-stranded and carries an amino acid.
• Each tRNA is highly specific to one type of amino acid.
• Aminoacyl-tRNA synthetases are the key to ensuring alanine binds only alanine-specific tRNA, attaching an amino acid to a tRNA terminal group to make an ester bond.
• The anticodon helps tRNA to align with the mRNA.
• The mRNA and tRNA are antiparallel at the point of contact.

Ensuring accurate translation

• There are two parts to ensure right amino acids are connected
• Pairing the anticodon to the codon
• The synthetase matching the tRNA to the correct acid

The genetic code

• The order of DNA bases dictates amino acid sequences in proteins; the nature of the code remained unknown.
• The code could not be one-to-one because there are 4 bases and 20 amino acids.
• Marshall Nirenberg made a synthetic mRNA consisting only of uracil bases, and put into a synthetic protein-producing system and found only phenylalanine was the result.
• Nirenberg determined that UUU coded for phenylalanine.
• By 1967, the three based genetic code was broken.
• Each amino acid is coded for by a sequence of three bases, called a codon
• Genetic code is nearly universal, with the same three bases coding for the same amino acid in almost every organism.
• The universality of the genetic code infers all living matter on Earth originated from one primordial organism
• The finding provides evidence supporting Darwin's theory of evolution.
• Exceptions to the genetic code occur in mitochondrial DNA.
• The 64 possible combinations of four bases into triplets allows all known codons to be deciphered.
• Three codons signal “stop”: UAA, UAG, and UGA, terminating protein synthesis.
• The remaining 61 codons code for one of 20 amino acids, causing overlap.
• Some amino acids have as many as six codons, for example Leucine, which has code UUA, UUG, CUU, CUC, CUA, and CUG.

Protein Synthesis Initiation and Termination

• AUG serves as an initiation and Met codon.
• Methionine is initially, but not always, added into all protein synthesis.
• The mRNA is read from 5’ → 3’ and the first amino acid to methionine linked to initial methionine becomes N-terminal end translated
• If AUGGGCCAA is the mRNA, AUG is one codon for the first amino acid, GGC is the second codon, and CAA is the thirst; there are no overlapping codons and no nucleotides are interspersed.

Protein Synthesis (Detailed Mechanism)

• Begins with activation, initiation, elongation, and termination.
• Molecular components take place in each step.
• Activation includes Amino acids, ATP, tRNAs, aminoacyl-tRNA synthetases.
• Initiation includes fMet-tRNAfMet, 30S ribosome, initiation factors, mRNA with Shine-Dalgarno sequence, 50S ribosome, GTP.
• Elongation: 30S, 50S ribosomes, aminoacyl-tRNAs, elongation factors, mRNA and GTP
• Termination includes Release factors and GTP

Activation (Protein Synthesis)

• Each amino acid gets activated by a reaction with an ATP molecule.
• This makes an aminoacyl-AMP.
• The second part transfers the activated ammino acid and binds to tRNA via Aminoacyl-tRNA Synthetase.
• Synthetases recognize amino acid substrates via nucleotide sequences on tRNA, referred to as the "second genetic code," ensuring correct pairing before opportunity to check later.
• Once amino acis is in tRNA it wont be checked so aminoacyl tRNA has to be correct.

Initiation of Protein Synthesis (Detailed)

• Synthesis begins with tRNAfMet, carrying formylated Met bonded to a 30S ribosomal body to make a pre-initiation complex
• Initiation factors aid translation.
• Pre-initiation complex binds the mRNA and the ribosome aligns via recognizing the Shine-Dalgarno sequence, where the fMet-tRNAfMet anticodon and UAC align against start codon AUG.
• 50S ribosomal body joins the 30S ribosomal complex now carrying three sites.

Elongation of peptide chain

• Only the tRNAs with anticondons that carry the corresponding mRNA can correspond with the help of elongation factors and GTP
• The new amino acid, alanine, is bonded to the Met by peptidyl transferase with the empy RNA remaining on P site
• Whole ribosome moves on from one codon of mRNA in a step known as translocation
• In the next stage to elongate, A site becomes associated with mRNA.
• The tRNA carries glycine and transferase establishes new peptide bond .
• Elongation occurs again

Ribosomes

• Model for translational process created with Cryo-electron microscopy and X-ray diffraction studies show that Elongation factor proteins fit the gap between 50S and 30S bodies
• At the P and A site, tRNAs occupy central cavity as orrange beads
• Peptide chain bond is done via nucleophilic attack by group and tRNA at p spot

Termination of Protein Synthesis

• The translocated codon becomes a stop and the polypeptide chains are ended
• Releasing factors separate factors from tRNA and everything is released from the Ribosomes/mRNA

Gene Regulation

• Genes are not active all the time through development but switched on and off that allows the specialization of cells.
• RNA/protein actions are not single for control but multiple, which is called gene regulation
• Eukaryotes have promoters, enhancers, and response elements

Promoters

• Promoters are an enzyme that defines a transciption site
• Has RNA enzyme thats known as TFII and letters for pol2 transctiption
• Factors bind the DNA for transcript action, initaiting the pre initial complex at phosphorylation stage of RNA Polymerase.
• Enhancers help control steady and normal level to vary by facotor of one million for Eukaryotic cells