Overview: The Flow of Genetic Information

¥     The information content of DNA is in the form of specific sequences of nucleotides

¥     The DNA inherited by an organism leads to specific traits by dictating the synthesis of proteins

¥     Proteins are the links between genotype and phenotype

¥     Gene expression, the process by which DNA directs protein synthesis, includes two stages: transcription and translation

 

Concept 17.1: Genes specify proteins via transcription and translation

¥     How was the fundamental relationship between genes and proteins discovered?

Evidence from the Study of Metabolic Defects

¥     In 1902, British physician Archibald Garrod first suggested that genes dictate phenotypes through enzymes that catalyze specific chemical reactions

¥     He thought symptoms of an inherited disease reflect an inability to synthesize a certain enzyme

¥     Linking genes to enzymes required understanding that cells synthesize and degrade molecules in a series of steps, a metabolic pathway

Nutritional Mutants in Neurospora: Scientific Inquiry

¥     George Beadle and Edward Tatum exposed bread mold to X-rays, creating mutants that were unable to survive on minimal media

¥     Using crosses, they and their coworkers identified three classes of arginine-deficient mutants, each lacking a different enzyme necessary for synthesizing arginine

¥     They developed a one gene–one enzyme hypothesis, which states that each gene dictates production of a specific enzyme

The Products of Gene Expression: A Developing Story

¥     Some proteins arenÕt enzymes, so researchers later revised the hypothesis: one gene–one protein

¥     Many proteins are composed of several polypeptides, each of which has its own gene

¥     Therefore, Beadle and TatumÕs hypothesis is now restated as the one gene–one polypeptide hypothesis

¥     Note that it is common to refer to gene products as proteins rather than polypeptides

Basic Principles of Transcription and Translation

¥     RNA is the bridge between genes and the proteins for which they code

¥     Transcription is the synthesis of RNA using information in DNA

¥     Transcription produces messenger RNA (mRNA)

¥     Translation is the synthesis of a polypeptide, using information in the mRNA

¥     Ribosomes are the sites of translation

¥     In prokaryotes, translation of mRNA can begin before transcription has finished

¥     In a eukaryotic cell, the nuclear envelope separates transcription from translation

¥     Eukaryotic RNA transcripts are modified through RNA processing to yield the finished mRNA

¥     A primary transcript is the initial RNA transcript from any gene prior to processing

¥     The central dogma is the concept that cells are governed by a cellular chain of command:  DNA ¨ RNA ¨ protein

The Genetic Code

¥     How are the instructions for assembling amino acids into proteins encoded into DNA?

¥     There are 20 amino acids, but there are only four nucleotide bases in DNA

¥     How many nucleotides correspond to an amino acid?

Codons: Triplets of Nucleotides

¥     The flow of information from gene to protein is based on a triplet code: a series of nonoverlapping, three-nucleotide words

¥     The words of a gene are transcribed into complementary nonoverlapping three-nucleotide words of mRNA

¥     These words are then translated into a chain of amino acids, forming a polypeptide

¥     During transcription, one of the two DNA strands, called the template strand, provides a template for ordering the sequence of complementary  nucleotides in an RNA transcript

¥     The template strand is always the same strand for a given gene

¥     During translation, the mRNA base triplets, called codons, are read in the 5¢ to 3¢ direction

¥     Codons along an mRNA molecule are read by translation machinery in the 5¢ to 3¢ direction

¥     Each codon specifies the amino acid (one of 20) to be placed at the corresponding position along a polypeptide

Cracking the Code

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

¥     Of the 64 triplets, 61 code for amino acids; 3 triplets are ÒstopÓ signals to end translation

¥     The genetic code is redundant (more than one codon may specify a particular amino acid) 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

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

 

Concept 17.2: Transcription is the DNA-directed synthesis of RNA: a closer look

¥     Transcription is the first stage of gene expression

Molecular Components of Transcription

¥     RNA synthesis is catalyzed by RNA polymerase, which pries the DNA strands apart and hooks together the RNA nucleotides

¥     The RNA is complementary to the DNA template strand

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

¥     The DNA sequence where RNA polymerase attaches is called the promoter; in bacteria, the sequence signaling the end of transcription is called the terminator

¥     The stretch of DNA that is transcribed is called a transcription unit

Synthesis of an RNA Transcript

¥     The three stages of transcription

   Initiation

   Elongation

   Termination

RNA Polymerase Binding and Initiation of Transcription

¥     Promoters signal the transcriptional start point and usually extend several dozen nucleotide pairs upstream of the start point

¥     Transcription factors mediate the binding of RNA polymerase and the initiation of transcription

¥     The completed assembly of transcription factors and RNA polymerase II bound to a promoter is called a transcription initiation complex

¥     A promoter called a TATA box is crucial in forming the initiation complex in eukaryotes

Elongation of the RNA Strand

¥     As RNA polymerase moves along the DNA, it untwists the double helix, 10 to 20 bases at a time

¥     Transcription progresses at a rate of 40 nucleotides per second in eukaryotes

¥     A gene can be transcribed simultaneously by several RNA polymerases

¥     Nucleotides are added to the 3¢ end of the growing RNA molecule

Termination of Transcription

¥     The mechanisms of termination are different in bacteria and eukaryotes

¥     In bacteria, the polymerase stops transcription at the end of the terminator and the mRNA can be translated without further modification

¥     In eukaryotes, RNA polymerase II transcribes the polyadenylation signal sequence; the RNA transcript is released 10–35 nucleotides past this polyadenylation sequence

 

Concept 17.3: Eukaryotic cells modify RNA after transcription

¥     Enzymes in the eukaryotic nucleus modify pre-mRNA (RNA processing) before the genetic messages are dispatched to the cytoplasm

¥     During RNA processing, both ends of the primary transcript are usually altered

¥     Also, usually some interior parts of the molecule are cut out, and the other parts spliced together

Alteration of mRNA Ends

¥     Each end of a pre-mRNA molecule is modified in a particular way

   The 5¢ end receives a modified nucleotide 5¢ cap

   The 3¢ end gets a poly-A tail

¥     These modifications share several functions

   They seem to facilitate the export of mRNA to the cytoplasm

   They protect mRNA from hydrolytic enzymes

   They help ribosomes attach to the 5¢ end

Split Genes and RNA Splicing

¥     Most eukaryotic genes and their RNA transcripts have long noncoding stretches of nucleotides that lie between coding regions

¥     These noncoding regions are called intervening sequences, or introns

¥     The other regions are called exons because they are eventually expressed, usually translated into amino acid sequences

¥     RNA splicing removes introns and joins exons, creating an mRNA molecule with a continuous coding sequence

¥     In some cases, RNA splicing is carried out by spliceosomes

¥     Spliceosomes consist of a variety of proteins and several small nuclear ribonucleoproteins (snRNPs) that recognize the splice sites

Ribozymes

¥     Ribozymes are catalytic RNA molecules that function as enzymes and can splice RNA

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

¥     Three properties of RNA enable it to function as an enzyme

   It can form a three-dimensional structure because of its ability to base-pair with itself

   Some bases in RNA contain functional groups that may participate in catalysis

   RNA may hydrogen-bond with other nucleic acid molecules

The Functional and Evolutionary Importance of Introns

¥     Some introns contain sequences that may regulate gene expression

¥     Some genes can encode more than one kind of polypeptide, depending on which segments are treated as exons during splicing

¥     This is called alternative RNA splicing

¥     Consequently, the number of different proteins an organism can produce is much greater than its number of genes

¥     Proteins often have a modular architecture consisting of discrete regions called domains

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

¥     Exon shuffling may result in the evolution of new proteins

 

Concept 17.4: Translation is the RNA-directed synthesis of a polypeptide: a closer look

¥     Genetic information flows from mRNA to protein through the process of translation

Molecular Components of Translation

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

¥     tRNAs transfer amino acids to the growing polypeptide in a ribosome

¥     Translation is a complex process in terms of its biochemistry and mechanics

The Structure and Function of Transfer RNA

¥     Molecules of tRNA are not identical

   Each carries a specific amino acid on one end

   Each has an anticodon on the other end; the anticodon base-pairs with a complementary codon on mRNA

¥     A tRNA molecule consists of a single RNA strand that is only about 80 nucleotides long

¥     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

¥     tRNA is roughly L-shaped

¥     Accurate translation requires two steps

   First: a correct match between a tRNA and an amino acid, done by the enzyme aminoacyl-tRNA synthetase

   Second: a correct match between the tRNA anticodon and an mRNA codon

¥     Flexible pairing at the third base of a codon is called wobble and allows some tRNAs to bind to more than one codon

Ribosomes

¥     Ribosomes facilitate specific coupling of tRNA anticodons with mRNA codons in protein synthesis

¥     The two ribosomal subunits (large and small) are made of proteins and ribosomal RNA (rRNA)

¥     Bacterial and eukaryotic ribosomes are somewhat similar but have significant differences: some antibiotic drugs specifically target bacterial ribosomes without harming eukaryotic ribosomes

¥     A ribosome has three binding sites for tRNA

   The P site holds the tRNA that carries the growing polypeptide chain

   The A site holds the tRNA that carries the next amino acid to be added to the chain

   The E site is the exit site, where discharged tRNAs leave the ribosome

Building a Polypeptide

¥     The three stages of translation

   Initiation

   Elongation

   Termination

¥     All three stages require protein ÒfactorsÓ that aid in the translation process

Ribosome Association and Initiation of Translation

¥     The initiation stage of translation brings together mRNA, a tRNA with the first amino acid, and the two ribosomal subunits

¥     First, a small ribosomal subunit binds with mRNA and a special initiator tRNA

¥     Then the small subunit moves along the mRNA until it reaches the start codon (AUG)

¥     Proteins called initiation factors bring in the large subunit that completes the translation initiation complex

Elongation of the Polypeptide Chain

¥     During the elongation stage, amino acids are added one by one to the preceding amino acid at the C-terminus of the growing chain

¥     Each addition involves proteins called elongation factors and occurs in three steps: codon recognition, peptide bond formation, and translocation

¥     Translation proceeds along the mRNA in a 5′ to 3′ direction

Termination of Translation

¥     Termination occurs when a stop codon in the mRNA reaches the A site of the ribosome

¥     The A site accepts a protein called a release factor

¥     The release factor causes the addition of a water molecule instead of an amino acid

¥     This reaction releases the polypeptide, and the translation assembly then comes apart

Polyribosomes

¥     A number of ribosomes can translate a single mRNA simultaneously, forming a polyribosome (or polysome)

¥     Polyribosomes enable a cell to make many copies of a polypeptide very quickly

Completing and Targeting the Functional Protein

¥     Often translation is not sufficient to make a functional protein

¥     Polypeptide chains are modified after translation or 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 its three-dimensional shape

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

¥     Some polypeptides are activated by enzymes that cleave them

¥     Other polypeptides come together to form the subunits of a protein

Targeting Polypeptides to Specific Locations

¥     Two populations of ribosomes are evident in cells: free ribsomes (in the cytosol) and bound ribosomes (attached to the ER)

¥     Free ribosomes mostly synthesize proteins that function in the cytosol

¥     Bound ribosomes make proteins of the endomembrane system and proteins that are secreted from the cell

¥     Ribosomes are identical and can switch from free to bound

¥     Polypeptide synthesis always begins in the cytosol

¥     Synthesis finishes in the cytosol unless the polypeptide signals the ribosome to attach to the ER

¥     Polypeptides destined for the ER or for secretion are marked by a signal peptide

¥     A signal-recognition particle (SRP) binds to the signal peptide

¥     The SRP brings the signal peptide and its ribosome to the ER

 

Concept 17.5: Mutations of one or a few nucleotides can affect protein structure and function

¥     Mutations are changes in the genetic material of a cell or virus

¥     Point mutations are chemical changes in just one base pair of a gene

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

Types of Small-Scale Mutations

¥     Point mutations within a gene can be divided into two general categories

   Nucleotide-pair substitutions

   One or more nucleotide-pair insertions or deletions

Substitutions

¥     A nucleotide-pair substitution replaces one nucleotide and its partner with another pair of nucleotides

¥     Silent mutations have no effect on the amino acid produced by a codon because of redundancy in the genetic code

¥     Missense mutations still code for an amino acid, but not the correct amino acid

¥     Nonsense mutations change an amino acid codon into a stop codon, nearly always leading to a nonfunctional protein

Insertions and Deletions

¥     Insertions and deletions are additions or losses of nucleotide pairs in a gene

¥     These mutations have a disastrous effect on the resulting protein more often than substitutions do

¥     Insertion or deletion of nucleotides may alter the reading frame, producing a frameshift mutation

Mutagens

¥     Spontaneous mutations can occur during DNA replication, recombination, or repair

¥     Mutagens are physical or chemical agents that can cause mutations

 

Concept 17.6: While gene expression differs among the domains of life, the concept of a gene is universal

¥     Archaea are prokaryotes, but share many features of gene expression with eukaryotes

Comparing Gene Expression in Bacteria, Archaea, and Eukarya

¥     Bacteria and eukarya differ in their RNA polymerases, termination of transcription, and ribosomes; archaea tend to resemble eukarya in these respects

¥     Bacteria can simultaneously transcribe and translate the same gene

¥     In eukarya, transcription and translation are separated by the nuclear envelope

¥     In archaea, transcription and translation are likely coupled

What Is a Gene? Revisiting the Question

¥     The idea of the gene has evolved through the history of genetics

¥     We have considered a gene as

   A discrete unit of inheritance

   A region of specific nucleotide sequence in a chromosome

   A DNA sequence that codes for a specific polypeptide chain

¥     In summary, a gene can be defined as a region of DNA that can be expressed to produce a final functional product, either a polypeptide or an RNA molecule