Alternative RNA Splicing & Exon Shuffling

• Occurence of introns means a single gene can encode multiple different polypeptides

Alternative RNA Splicing

• Different segments are treated as exons during RNA processing
• The Human Genome Project suggests alternative RNA splicing is why humans have similar number of genes to nematodes (roundworms)
• Alternative splicing results in an organism producing more protein products than it has genes

Domains

• Proteins often have a modular architecture with discrete structural and functional regions
• One domain of an enzyme may include the active site
• Others allow the enzyme to bind to a cellular membrane
• Different exons code for different protein domains

Exon Shuffling

• A process where the presence of introns in a gene may facilitate beneficial protein evolution
• Introns increase the probability of crossing over between exons of alleles, providing more terrain for crossovers without interrupting coding sequences
• Exon shuffling can create new exon combinations and proteins with altered structure and function
• It could also lead to mixing and matching exons between completely different genes
• This can create new proteins with novel function combinations leading to beneficial variations

Concept Check 17.3

• Human cells make 75,000-100,000 different proteins from 20,000 protein-coding genes through alternative RNA splicing
• Conceptually, RNA splicing can be compared to watching a pre-recorded TV show
• In this analogy, Introns would correspond to parts cut from the show

Concept 17.4 Translation

• Genetic information flows from mRNA to protein through translation
• Focus on the basic steps of translation in bacteria and eukaryotes, highlighting key differences

Translation Basics

• mRNA moves through a ribosome and codons are translated into amino acids
• tRNA molecules are translators with a specific anticodon on one end and a corresponding amino acid on the other
• A tRNA adds its amino acid cargo to a growing polypeptide chain when the anticodon hydrogen-bonds to the complementary codon on the mRNA

Molecular Components of Translation

• During translation, a cell "reads" a genetic message and builds a polypeptide
• The message is a series of codons along an mRNA molecule
• The translator is a transfer RNA (tRNA)
• The function of tRNA is to transfer amino acids from cytoplasm to polypeptide in ribosome
• Cytoplasm contains all 20 amino acids, synthesized, or taken from surrounding solution
• Ribosome adds each amino acid to the growing end of a polypeptide chain

* Transfer RNA Structure and Function

• Each tRNA molecule translates an mRNA codon into a specific amino acid
• A tRNA molecule is a single RNA strand of ~80 nucleotides
• Self complementary nucleotide base pairing allows the strand to fold back on itself, creating a particular 3D structure
• Flattened, a tRNA molecule looks like a cloverleaf
• It twists and folds into a compact, L-shaped 3D structure with the 5' and 3' ends of the linear tRNA located near one end
• The protruding 3' end acts as the attachment site for an amino acid
• The loop extending from the other end includes the anticodon, the nucleotide triplet that base-pairs to a specific mRNA codon
• Anticodons are conventionally written 3' → 5' to align properly with codons written 5'→ 3'
• tRNA base-pairs with with codon by hydrogen bonding.

* tRNA Molecule as Translator

• tRNA in the context of a ribosome, translates a nucleic acid word (mRNA codon) to a protein word (amino acid)
• Like mRNA, tRNA molecules are transcribed from DNA templates
• Eukaryotic cells create tRNA in the nucleus and it travels to the cytoplasm to participate in translation
• Each tRNA molecule is used repeatedly in both bacteria and eukaryotic cells
• tRNA picks up amino acid in the cytosol, deposits it onto a polypeptide chain at the ribosome, then leaves to pick up the same amino acid

* Accuracy of Translation

• Accurate translation requires two instances of molecular recognition
• First, tRNA that binds to an mRNA codon specifying a particular amino acid must carry that amino acid and no other to the ribosome
• tRNAs and amino acids are matched through aminoacyl-tRNA synthetases
• Each aminoacyl-tRNA synthetase's active site fits only a combination of amino acid and tRNA, ensured by the structure of the amino acid attachment and anticodon ends
• There are 20 synthetases, one for each amino acid
• Synthetase catalyzes the covalent attachment of the amino acid to its appropriate tRNA, driven by ATP hydrolysis
• The resulting aminoacyl tRNA, or charged tRNA, is released and is able to deliver its amino acid to a growing polypeptide chain on a ribosome
• The second instance of molecular recognition is pairing the tRNA anticodon with the appropriate mRNA codon
• If each mRNA codon had one tRNA type, there would be 61 tRNAs, but bacteria only have 45
• Some tRNA are able to bind to more than one codon.
• Bacteria can achieve this because the rules for base pairing between the third nucleotide base of a codon and the corresponding base of a tRNA anticodon are less strict
• The nucleotide base U at the 5' end of a tRNA anticodon can pair with either A or G in the third position (at the 3' end) of an mRNA codon, called wobble
• Wobble explains why synchronous codons for a given amino acid most often differ in their third nucleotide base
• A tRNA with the anticodon 3'-UCU-5' can base-pair with either the mRNA codon 5'-AGA-3' or 5'-AGG-3', both code for arginine
• degenerate: One aminoacid can be coded with more than one codon.
• unmbiguous: Each codon is related to one amino acid.

* Structure and Function of Ribosomes

• Ribosomes of bacteria and eukaryotes are similar in structure and function
• Eukaryotic ribosomes are slightly larger and differ somewhat in their molecular composition
• Differences are medically significant with antibiotic drugs being able to inactivate bacterial ribosomes without affecting eukaryotic ribosomes
• A ribosome has a large and small subunit, One-third of the ribosome’s mass is made of proteins, and the rest consists of three rRNAs (bacteria) or four (eukaryotes)
• Most cells have thousands of ribosomes, rRNA is the most abundant cellular RNA
• In eukaryotes, ribosome subunits are made in the nucleolus. Ribosomal RNA genes are transcribed, and the RNA is processed and assembled with proteins
• Completed Subunits export to cytoplasm.
• Structure reflects its function of bringing an mRNA plus tRNAs carrying amino acids
• The mRNA has a binding site for the ribosome
• Ribosome has a binding site for mRNA and three tRNA binding sites; the P, A, and E sites
• The P site (peptidyl-tRNA binding site) holds the tRNA carrying the growing polypeptide chain
• A site (aminoacyl-tRNA binding site) holds the tRNA carrying the next amino acid
• Discharged tRNAs leave from the E site (exit site)
• It catalyzes peptide bond formation, and the polypeptide passes through an exit tunnel
• Accepted model is that rRNAs, not ribosomal proteins, are primarily responsible for the structure and function of the ribosome
• Ribosomal proteins support the shape changes of the rRNA molecules as they carry out catalysis during translation
• rRNA is the mail construct of A and P sites and it acts as catalyst of peptide bond formation
• A ribosome is actually one colossal ribozyme

Building a Polypeptide

• Translation, the synthesis of a polypeptide, has three stages; initiation, elongation, and termination
• All require protein "factors" that aid in translation and energy, provided by GTP hydrolysis

* Ribosome Association and Initiation of Translation

• During initiation, mRNA, tRNA bearing the first amino acid, and the two subunits of a ribosome join
• In bacteria, the small subunit binds the mRNA at a specific RNA sequence prior to the start codon, AUG, and then attach to initiator tRNA.
• In eukaryotes, the small subunit and initiator tRNA already bounded, bind to the 5' cap of mRNA
• The initiator tRNA finds AUG and hydrogen bonds
• This binding establishes a codon reading frame for mRNA.
• next step is the attachment of larger ribosomal subunit.
• proteins called initiation factors bring all these components together
• Completing this initiation complex costs GTP for energy
• The initiator tRNA sits in the P site, and the A site is ready for the next tRNA
• A polypeptide is always synthesized in one direction, from the initial methionine at the amino end (N-terminus) to the final amino acid at the carboxyl end (C-terminus)

* Elongation of the Polypeptide Chain

• Elongation adds amino acids one by one to the previous amino acid at the C-terminus
• Each addition costs multiple elongation factors and consists of three-step cycle:

1. Codon recognition ( requires GTP increasing accuracy and efficiency )
2. Peptide bond formation
3. Translocation ( the movement of ribosome + emptying E site which requires GTP )

• Elongation takes less than a tenth of a second in bacteria
• Empty tRNAs released return to the cytoplasm to be reloaded

Termination of Translation

• Termination is the final stage of translation
• Elongation continues untill at stop codon in mRNA
• The nucleotide base triplets UAG, UAA, and UGA (all written 5'→ 3') do not code for amino acids, they signal termination
• A release factor, a protein shaped like an aminoacyl tRNA, binds directly to the stop codon in the A site
• This causes the addition of a water molecule instead of an amino acid to the polypeptide chain, splitting the polypeptide and releases the chain
• Breakdown costs two GTP molecules
• Uncharged aminoacid release from P site

Completing and Targeting the Functional Protein

• The process of translation is often insufficient to make a functional protein
• Polypeptide chains undergo modifications, as well used target completed proteins to specific sites in the cell

Protein Folding and Post-Translational Modifications

• During synthesis, polypeptide chain coils and folds spontaneously via amino acid sequence, forming a protein with a specific three-dimensional structure
• A gene determines primary structure, which in turn determines shape

Protein Modification

• Additional steps of post-translational modifications may be required before the protein can begin functioning within the cell
• Certain amino acids may be chemically modified by adding sugars, lipids, phosphate groups, or other additions
• Enzymes may remove amino acids from the leading amino end of the polypeptide chain
• Polypeptide chains may be cleaved into 2+ pieces
• Multiple separately synthesized polypeptides can combine if the protein has quaternary structure

Targeting Polypeptides to Specific Locations

• Active eukaryotic cells contain evident, free and bound ribosomes
• Free ribosomes are suspended in the cytosol and mainly synthesize proteins that stay and function there
• Bound ribosomes attach to the cytosolic side of the endoplasmic reticulum (ER) or to the nuclear envelope, creating proteins of the endomembrane system
• Note ribosomes are identical and can alternate between being free ribosomes or bound
• Polypeptide synthesis begins in the cytosol as a free ribosome starts to translate an mRNA molecule, unless the potein needs an ER signal
• Polypeptides destined for the endomembrane system are marked by a signal peptide, which targets the protein to the ER
• These polypeptides are recognized when the structure causes the signal is recognized by signal-recognition particle (SRP), a protein-RNA complex
• Complex proceeds to a receptor within the ER for continuation
• Polypeptide then snakes through the membrane into an ER lumen
• Signal peptides may be cleaved, or remain embedded depending on the final result
• Transports to destination within a vesicle
• All signal peptides are used to target polypeptides to different cellular environments

Making Multiple Polypeptides

• A single polypeptide is synthesized using mRNA
• Many copies are required with multiple ribosomes
• Multiple ribosomes simultaneously translate an mRNA at given point
• Once a ribosome is far enough past the start codon, a second binds to the mRNA
• This results in strings of ribosomes, called polyribosomes (or polysomes)
• They enable rapid production of many polypeptide copies
• Bacteria and eukaryotes both increase the number of copies of a polypeptide by transcribing multiple mRNAs from the same gene
• The coordinate processes of transcription and translation differ between Bacteria and Eukaryotes due to organization

Bacterial vs Eukaryotic Coordination

• Bacteria lack compartmental organization, streamlined by coupling, and simultaneous transcription and translation of the same gene
• Newly made proteins in can quickly diffuse to function
• Eukaryotic cells segregate these processes by a nuclear envelope, allowing for expansive RNA processing

Coupled Transcription and Translation in Bacteria

• In bacterial cells, mRNA can begin translation when leading end peels away from the DNA template.
• In eukaryotic cells, mRNAs have been found to have a circular arrangement in which proteins hold the poly-A tail near the 5' cap, increasing translation efficiency.

Concept Check 17.4

• Two processes that ensure the correct amino acid is added to a growing polypeptide chain are tRNA matching via synthetases and stop codons
• A polypeptide to be secreted to reaches the endomembrane system by signal recognition particles