Gene Expression PDF

Figure 17.1b CONCEPT 17.1: Genes specify proteins via transcription and translation ====================================================================== - The DNA inherited by an organism leads to specific traits by dictating the synthesis of proteins - Proteins are the links between ge...

Figure 17.1b CONCEPT 17.1: Genes specify proteins via transcription and translation ====================================================================== - 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 - How was the relationship between proteins and DNA discovered? Basic Principles of Transcription and Translation ================================================= - RNA is the bridge between genes and protein synthesis - **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 Figure 17.4 - 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, threenucleotide 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 Figure 17.5 - The template strand is always the same strand for a given gene - However, further along the chromosome, the opposite strand may be the template strand for a different gene Specific DNA sequences associated with the gene direct which strand is used as the template - The mRNA molecule produced is complementary to the template strand - During translation, the mRNA base triplets, called **codons**, are read in the 5′ → 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 - 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 Figure 17.6 CONCEPT 17.2: Transcription is the DNAdirected 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 joins together the RNA nucleotides - The RNA is complementary to the DNA template strand - RNA polymerase does not need any primer - RNA synthesis follows the same base-pairing rules as DNA, except that uracil substitutes for thymine Figure 17.8 - 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 transcription **start point** and usually extend several dozen nucleotide pairs upstream of the start point - **Transcription factors** help guide 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 Figure 17.9 Elongation of the RNA Strand ---------------------------- - As RNA polymerase moves along the DNA, it untwists the double helix, 10--20 nucleotides at a time - Nucleotides are added to the 3′ end of the growing RNA molecule - Transcription progresses at a rate of 40 nucleotides per second in eukaryotes - A gene can be transcribed simultaneously by several RNA polymerases Figure 17.10 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 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 altered - Also, in most cases, certain interior sections of the molecule are cut out and the remaining 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 Figure 17.11 Split Genes and RNA Splicing ============================ - Most eukaryotic genes and their RNA transcripts have long noncoding stretches of nucleotides that lie between coding regions - These are removed through **RNA splicing** - The noncoding segments in a gene are called intervening sequences, or **introns** - The other regions are called **exons** because they are eventually expressed, usually translated into amino acid sequences Figure 17.12 - The removal of introns is accomplished by spliceosomes - **Spliceosomes** consist of a variety of proteins and several small RNAs that recognize the splice sites - The RNAs of the spliceosome also catalyze the splicing reaction Figure 17.13 Ribozymes --------- - **Ribozymes** are catalytic RNA molecules that function as enzymes and can splice RNA - 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 regulate gene expression and many affect gene products - 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** - 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 by mixing and matching exons between different genes Figure 17.14 CONCEPT 17.4: Translation is the RNAdirected synthesis of a polypeptide: *a closer look* ======================================================================================== 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 Figure 17.15 The Structure and Function of Transfer RNA ------------------------------------------ - Each tRNA molecule enables translation of a given mRNA codon into a certain amino acid - Each carries a specific amino acid on one end - Each has an **anticodon** on the other end; the anticodon basepairs 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 with the 5′ and 3′ ends both located near one end of the structure - The protruding 3′ end acts as an attachment site for an amino acid Figure 17.16 - Accurate translation requires two instances of molecular recognition - 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 Figure 17.17 The Structure and Function of Ribosomes --------------------------------------- - Ribosomes facilitate specific coupling of tRNA anticodons with mRNA codons in protein synthesis - Eukaryotic ribosomes are somewhat larger than bacterial ribosomes and differ in their molecular composition - Some antibiotic drugs specifically inactivate bacterial ribosomes without harming eukaryotic ribosomes - The two ribosomal subunits (large and small) are made of proteins and **ribosomal RNAs (rRNAs)** - 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 Figure 17.18 Building a Polypeptide ====================== - The three stages of translation: - Initiation - Elongation - Termination - All three stages require protein "factors" that aid in the translation process - Energy is required for some steps, too Ribosome Association and Initiation of Translation -------------------------------------------------- - The initiation of translation starts when the small ribosomal subunit binds with mRNA and a special initiator tRNA - The initiator tRNA carries the amino acid methionine - 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 Figure 17.19 Elongation of the Polypeptide Chain ----------------------------------- - During elongation, amino acids are added one by one to the C-terminus of the growing chain - Each addition involves proteins called elongation factors - Elongation occurs in three steps: codon recognition, peptide bond formation, and translocation - Energy expenditure occurs in the first and third steps - Translation proceeds along the mRNA in a 5′ → 3′ direction - The ribosome and mRNA move relative to each other, codon by codon - The elongation cycles takes less than a tenth of a second in bacteria - Empty tRNAs released from the E site return to the cytoplasm, where they will be reloaded with the appropriate amino acid Figure 17.20 Termination of Translation -------------------------- - Elongation continues until a stop codon in the mRNA reaches the A site - 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 comes apart Figure 17.21 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 synthesis, a polypeptide chain begins to coil and fold spontaneously into a specific shape: a threedimensional molecule with secondary and tertiary structure - A gene determines the primary structure, and the primary structure in turn determines shape - Post-translational modifications may be required before the protein can begin doing its particular job in the cell Targeting Polypeptides to Specific Locations -------------------------------------------- - Two populations of ribosomes are evident in cells: free ribosomes (in the cytosol) and bound ribosomes - 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** - The signal peptide is a sequence of about 20 amino acids at or near the leading end of the polypeptide - A **signal-recognition particle (SRP)** binds to the signal peptide - The SRP escorts the ribosome to a receptor protein built into the ER membrane - The signal peptide is removed by an enzyme - Other kinds of signal peptides target polypeptides to other organelles Figure 17.22 Making Multiple Polypeptides in Bacteria and Eukaryotes ======================================================= - Multiple 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 Figure 17.23 - A bacterial cell ensures a streamlined process by coupling transcription and translation - In this case the newly made protein can quickly diffuse to its site of function Figure 17.24 - In eukaryotes, the nuclear envelope separates the processes of transcription and translation - RNA undergoes processing before leaving the nucleus Figure 17.25 CONCEPT 17.5: Mutations of one or a few nucleotides can affect protein structure and function ============================================================================================= - **Mutations** are changes in the genetic information of a cell - **Point mutations** are changes in just one nucleotide pair of a gene - The change of a single nucleotide in a DNA template strand can lead to the production of an abnormal protein - If a mutation has an adverse effect on the phenotype of the organism, the condition is referred to as a genetic disorder or hereditary disease Figure 17.26 Types of Small-Scale Mutations ============================== - Point mutations within a gene can be divided into two general categories: - Single nucleotide-pair substitutions - 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; most lead to a nonfunctional protein Figure 17.27a 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** - Insertions or deletions outside the coding part of a gene could affect how the gene is expressed Figure 17.27b New Mutations and Mutagens ========================== - Spontaneous mutations can occur during errors in DNA replication or recombination - **Mutagens** are physical or chemical agents that can cause mutations - Chemical mutagens fall into a variety of categories - Most carcinogens (cancer-causing chemicals) are mutagens, and most mutagens are carcinogenic 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 - A gene can be defined as a region of DNA that can be expressed to produce a final functional product that is