17.1 Genes Specify Proteins via Transcription and Translation

Overview

• The information found in DNA takes the form of specific nucleotide sequences

• Inherited DNA creates specific traits by regulating protein synthesis of proteins

• Gene expression - the process by which DNA directs the synthesis of proteins; Includes two stages: transcription and translation

• The ribosome is part of the cellular machinery for translation, AKA polypeptide synthesis

Archibald Garrod, a British Physician

• In 1902, Archibald Garrod suggested that genes

  dictate phenotypes through enzymes (proteins that catalyze a specific chemical reaction)

• Garrod said symptoms of an inherited disease reflect an inability to synthesize a certain enzyme

Nutritional Mutants in Neurospora: Scientific Inquiry

• Beadle and Tatum exposed bread mold to X-rays, creating mutants. Mutants couldn’t survive on minimal food due to the inability to synthesize certain molecules. Each mutant lacked a different enzyme. Beadle and Tatum then developed a one gene-one enzyme hypothesis (the hypothesis that a gene dictates the production of a specific enzyme)

• Cell synthesize and degrade molecules in a series of steps called a metabolic pathway

• Some proteins aren’t enzymes, so researchers later renamed the hypothesis as one gene-one protein hypothesis.

• Many proteins are composed of several polypeptides, each of which has its own gene. Therefore, Beadle and Tatum’s hypothesis is now restated as one gene-one polypeptide hypothesis (the hypothesis that a gene dictates the production of a specific polypeptide)

• Genome - All the genes for a certain species

• Proteome - Collection of all the proteins used in a species

Basic Principles of Transcription and Translation (Protein Synthesis)

• Transcription - the synthesis of any kind of RNA using a DNA template

• Messenger RNA (mRNA) - A type of RNA that carries a genetic message from DNA to ribosomes

• Translation - The synthesis of a polypeptide using the info in mRNA. There is a change of “language” from nucleotides to amino acids. Requires tRNA and takes place on ribosomes.

• Ribosomes - The site of protein synthesis.

Prokaryote vs Eukaryote

• Location of transcription: In the nucleus of eukaryotes and the cytoplasm of prokaryotes

• Transcription & Translation:

  • In prokaryotes, mRNA is immediately transcribed & translated without more processing (no cap, no poly-A tail, and no intron removal)

  • In eukaryotes, transcription and translation are separated by the nuclear envelope. Processing and modifications of pre-mRNA result in mRNA

• Primary Transcript - An initial RNA transcript from any gene; also called pre-mRNA when transcribed from a protein-coding gene

• Central Dogma - the idea that the flow of information went only one way

The Genetic Code

Codons: Triplets of Bases

• The flow of information from gene to protein is based on a triplet code (a series of non-overlapping, three-nucleotide code words that specify a sequence of amino acids for a polypeptide chain)

• Genes determine the sequence of nucleotide bases

• There are two DNA strands per gene. Only one is transcribed.

  • During transcription, a DNA strand called the template strand provides a pattern for ordering the sequence of nucleotides in an RNA transcript

• Codons - the basic unit of the genetic code; a three-nucleotide sequence of DNA or mRNA that codes for a specific amino acid

• During translation, the mRNA codons, are read in the 5’ to 3’ direction

• Coding Strand - the nontemplate DNA strand, which has the same sequence as the mRNA except it has thymine (T) instead of uracil (U)

Cracking the Code

• All 64 codons were deciphered by the mid-1960s

• The genetic code is redundant but not ambiguous; no codon specifies more than one amino acid

• Codons must be read in the correct reading frame (correct groupings) in order for the specified polypeptide to be produced

  • Frameshifts can be problematic

  • If the frameshift occurs in an intron, then it does not make a difference

Evolution of the Genetic Code

• The genetic code is nearly universal, shared by the simplest bacteria to the most complex animals

• Genes can be transcribed and translated after being transplanted from one species to another

17.2 Transcription: Its Components and Stages

Molecular Components of Transcription

• RNA polymerase - An enzyme that catalyzes the synthesis of RNA; it pries the DNA strands apart and hooks together the RNA nucleotides

  • RNA synthesis follows the same base-pairing rules as DNA, except uracil substitutes for thymine

• Promoter - The DNA sequence where RNA polymerase attaches and transcription (RNA synthesis) is initiated

  • RNA polymerase II - one of three eukaryotic RNA polymerase that is used for pre-mRNA synthesis (prokaryotes have only ONE type of RNA polymerase)

• Terminator - In prokaryotes, a sequence that signals the end of transcription

  • Eukaryotes don’t have a terminator

• Transcription Unit - A region of DNA that is transcribed into an RNA molecule; Requires modifications only in eukaryotes

• The three stages of transcription are: initiation, elongation, and termination

Stage 1 - Initiation & RNA Polymerase Binding

• Start Point - In transcription, the nucleotide position on the promotor where RNA polymerase begins transcription

• The DNA template strand (direction and location of transcription) is determined by the location and orientation of RNA polymerase binding on the promotor

• Transcription Factors - In eukaryotes, a group of regulatory proteins that mediate the binding of RNA polymerase and the initiation of transcription

• Transcription Initiation Complex - the complete assembly of transcription factors and RNA polymerase II bound to a promoter

• TATA Box - In eukaryotes, a promoter DNA sequence that is crucial in the formation of the transcription initiation complex

• Summary: RNA polymerase & transcription factors bind to the promoter. This signals the DNA to unwind so the enzyme can ''read'' the bases in the template strand. The enzyme is now ready to make a strand of mRNA with a complementary sequence of bases.

Stage 2 - Elongation of the RNA Strand

• During elongation, RNA polymerase moves along the DNA. It untwists the double helix, exposing 10 to 20 bases at a time, and adds a matching RNA nucleotide for each nucleotide in the template.

• Transcription progresses at a rate of 40 nucleotides per/sec in eukaryotes

• A gene can be transcribed simultaneously by several RNA polymerases

Stage 3 - Termination of Transcription

• The mechanisms of termination are different in prokaryotes and eukaryotes

• In prokaryotes, the polymerase stops transcription at the end of the terminator

• In eukaryotes, RNA polymerase II continues transcription after the pre-mRNA is cleaved from the growing RNA chain; the polymerase eventually falls off the DNA

17.3 Eukaryotic Cells Modify RNA after Transcription

• RNA Processing - Modification of the primary transcript (pre-mRNA) in the nucleus by enzymes before the genetic message is sent to the cytoplasm; Includes RNA splicing (removal of introns & joining of exons), and modification of the 5’ and 3’ ends

• RNA processing produces an mRNA molecule ready for translation

Alteration/Modification of pre-mRNA Ends

• During RNA processing, each end of the primary transcript (pre-mRNA) is modified.

  • The 5’ end receives a 5’ cap (a modified form of guanine nucleotide)

  • The 3’ end receives a poly-A tail (a sequence of 50-250 adenine nucleotides)

• These modifications share three functions

  • Facilitate the export of mRNA from the nucleus

  • Protect mRNA from degradation by hydrolytic enzymes

  • Help ribosomes attach to the 5’ end of the mRNA

Split Genes and RNA Splicing

• RNA Splicing - The stage of RNA processing that involves the removal of introns and the joining of exons, making a continuous sequence

• Introns - the noncoding nucleotide segments of eukaryotic genes and their RNA transcripts that lie between coding regions

• Exons - the nucleotide segments of eukaryotic genes and their RNA transcripts that are eventually expressed, usually translated into amino acid sequences

• In some cases, RNA splicing is carried out by spliceosomes (a large complex made of proteins and several small nuclear ribonucleoproteins (snRNPs) that recognize the splice sites)

Ribozymes

• Ribozymes - RNA molecules that function as enzymes and can splice RNA

• The discovery of ribozymes rendered obsolete the belief that all biological catalysts were proteins

The Functional and Evolutionary Importance of Introns

• Alternative RNA Splicing - A type of eukaryotic gene regulation in which some genes can encode more than one kind of polypeptide, depending on which segments are treated as exons during RNA splicing

• The number of different proteins an organism can produce is much greater than its number of genes because of alternative splicing

• Proteins often have a modular architecture consisting of domains (discrete structural and functional regions)

• In many cases, different exons code for the different domains in a protein

17.4 Translation: Its Components and Stages

Molecular Components of Translation

• A cell translates an mRNA message into proteins with the help of transfer RNA (tRNA)

• Transfer RNA (tRNA) - An RNA molecule that is responsible for translating nucleotides to amino acids by transferring an amino acid to a growing polypeptide in a ribosome

• Molecules of tRNA are not identical

  • Each tRNA molecule enables the translation of a given mRNA codon into a certain amino acid

Structure of Transfer RNA (tRNA)

• The Parts of tRNA Molecule: a single RNA strand that is about 80 nucleotides long; Includes a specific amino acid on one end and an anticodon on the other end.

  • Anticodon - Nucleotide triplet at one end of a tRNA molecule that base-pairs with a complementary codon on mRNA

• The Shape of a tRNA Molecule: 3D and roughly L-shaped; When flattened into one plane to reveal its base pairing, a tRNA molecule looks like a cloverleaf

  • Because of hydrogen bonds, tRNA actually twists and folds into a three-dimensional molecule

• Accurate translation of a genetic message requires two steps:

  • First → A correct match between tRNA and amino acid; They are joined by aminoacyl-tRNA synthetase

  • Second → A correct match between the tRNA anticodon and an mRNA codon

• Aminoacyl-tRNA synthetases - An enzyme that joins each amino acid to the appropriate tRNA; There are 20 different synthetases, one for each amino acid.

• Wobble - Flexibility in the base-pairing rules in which the nucleotide at the 5’ end of a tRNA anticodon can form hydrogen bonds with more than one kind of base in the third position (3’ end) of a codon

Structure and Function of Ribosomes

Structure

• Contain two subunits (small and large), each consisting of proteins and ribosomal RNA (rRNA), and made in the nucleolus

  • Ribosomal RNA (rRNA) - RNA that joins with proteins to make ribosomes; the most abundant type of RNA

• Ribosomes have one binding site for mRNA and three binding sites for tRNA.

  • P Site - Holds the tRNA that carries the growing polypeptide chain

  • A Site - Holds the tRNA that carries the next amino acid to be added to the chain

  • E Site - The exit site, where discharged tRNAs leave the ribosome

Functions

• Ribosomes are the sites of protein synthesis

• Facilitate specific coupling of tRNA anticodons with mRNA codons in protein synthesis

3 Stages of Translation: Building a Polypeptide

Like transcription, the three stages of translation are initiation, elongation, and termination. All three stages require “protein factors” that offer support

Stage 1 - Initiation & Ribosome Association

• A ribosomal subunit binds with mRNA and a tRNA that holds methionine. The subunit moves along the mRNA until it reaches the start codon (AUG). A large ribosomal subunit is finally attached.

• All these complexes are brought together by initiation factors

• The complete complex of all these structures is called the translation initiation complex

Stage 2 - Elongation of the Polypeptide Chain

• During elongation, amino acids are added one by one to the preceding amino acid

• Each addition involves proteins called elongation factors and occurs in three steps: codon recognition, peptide bonding, and translocation

Stage 3 - Termination of Translation

• Termination occurs when a stop codon in the mRNA reaches the A site of the ribosome, then it binds with a release factor, causing the release of a polypeptide and the destruction of the translation assembly

• Release factor - A protein shaped like an aminoacyl tRNA; It binds directly to the stop codon in the A site, causing the addition of a water molecule instead of an amino acid

Completing and Targeting the Functional Protein

• Often translation is not sufficient to make a functional protein

• Polypeptide chains are modified after translation

• Completed proteins are targeted to specific sites in the cell

Protein Folding and Post-Translational Modifications

• During and after synthesis, a polypeptide chain spontaneously coils and folds into a three-dimensional shape

• Proteins may also require post-translational modifications before doing their job

Targeting Polypeptides to Specific Locations

• There are two types of ribosomes (free and bound). Both types are structurally and functionally identical. Both can swap places.

• Free Ribosomes - Ribosomes that are found in the cytosol; They synthesize proteins that function in the cytosol.

• Bound Ribosomes - Ribosomes that are attached to the endoplasmic reticulum (ER); They synthesize secretory proteins and proteins of the endomembrane system

• What determines whether a ribosome is free or bound? → Polypeptide synthesis starts with free ribosomes. A free ribosome becomes bound when the growing polypeptide cues the ribosome to attach to the ER.

• Polypeptides destined for the endoplasmic reticulum or for secretion are marked by a signal peptide (a sequence of amino acids that target a polypeptide to the endoplasmic reticulum or other organelles)

• Signal-Recognition Particle (SRP) - A protein RNA that recognizes and binds to the signal peptide, bringing it and its ribosome to the ER

Making Multiple Polypeptides in Bacteria and Eukaryotes

• Both prokaryotes and eukaryotes can make multiple polypeptides via the following two methods:

  • Transcribing multiple mRNAs from the same gene

  • Multiple ribosomes translating the same mRNA, forming a polyribosome/polysome

    • Polyribosomes/Polysomes - A group of several ribosomes attached to, and translating the same messenger RNA molecule, producing multiple polypeptides

17.5 Point Mutations Can Affect Protein Structure and Function

• Mutation - A change in the genetic material of a cell or virus; Source of the diversity of genes

• In terms of their effects, mutations can be beneficial, harmful or neutral

• The change of a single nucleotide in a DNA template strand can lead to the production of an abnormal protein

• Point Mutation - Small-scale change in a single nucleotide pair of a gene

• Genetic Disorder/Hereditary Disease - A mutation resulting in an adverse effect on the phenotype of a person

Types of Point Mutations

There are two main types of point mutations: base-pair substitutions and base-pair insertions or deletions

Base-Pair Substitutions

• Nucleotide-pair substitution - a type of point mutation in which one nucleotide pair is replaced by another pair

  • Silent Mutation - A nucleotide-pair substitution that has no observable effect on the phenotype

  • Missense Mutations - A nucleotide-pair substitution that results in a codon that codes for a different amino acid, but not necessarily the right amino acid

  • Nonsense Mutation - A nucleotide-pair substitution that changes an amino acid codon into a stop codon, resulting in a nonfunctional protein

• Base-pair substitution can cause missense or nonsense mutations

• Which is more common, missense mutations or nonsense mutations? → Missense

Base-Pair Insertions and Deletions

• Insertions - A mutation involving the addition of one or more nucleotide pairs to a gene

• Deletions - A mutation involving the loss of one or more nucleotide pairs from a gene

• Which types of point mutations are most dangerous? → Insertions and Deletions

• Frameshift Mutations - Insertion or deletion of nucleotides that alter the reading frame of the genetic message

New Mutations & Mutagens

• Spontaneous Mutations - A mutation caused when a DNA error isn’t fixed; Occur during replication, recombination, or repair

• Mutagens - Physical or chemical agents that interact with DNA and can cause mutations

  • Examples of mutagens: UV radiation, mercury, radon, lead, pesticide

Extra Vocab

• Gene Editing - Altering genes in a specific, predictable way

• CRISPR-Cas9 System is a technique for editing genes in living cells, involving a bacterial protein called Cas9 associated with a guide RNA complementary to a gene sequence of interest