Chapter 17: From Gene to Protein

17.1: Genes specify proteins via transcription and translation

Evidence from the Study of Metabolic Defects

· Gene expression = process by which DNA directs synthesis of proteins

· Garrod = first to suggest that genes dictate phenotypes through enzymes that catalyze specific chemical reactions in the cell

· Suggested symptoms of a disease reflect inability to make an enzyme

· Recognized that Mendel’s principles of heredity apply to humans

· Nutritional Mutants in Neurospora: Scientific Inquiry

· Beadle & Tatum worked with bread mold, bombarding it with X-rays to show genetic changes

· Identified that mutants couldn’t survive on minimal medium because they couldn’t synthesize certain molecules from medium

· needed to grow on complete growth medium that supplemented needed nutrients

· Distributed samples from mutant into different vials containing minimal medium plus an extra nutrient

· Supplement that allowed growth indicated metabolic defect

· Srd & Horowitz pinned down each mutant’s defect realizing that mutants in each class required a different set of compounds along the pathway which has three steps

· Suggests that each class was blocked at a different step

· Saw that since each mutant was defective in a single gene, it supported one gene one enzyme hypothesis that the function of a gene dictates the production of a specific enzyme

· The Products of Gene Expression: A Developing Story

· Not all proteins are enzymes one gene one protein

· But many proteins are constructed are constructed from two or more polypeptide chains, each chain of which is specified by its own gene

· One gene one polypeptide hypothesis

· Not totally accurate, many eukaryotic genes can each code for a set of closely related polypeptides via alternative splicing or code for RNA

Basic Principles of Transcription and Translation

· A gene doesn’t build a protein directly- nucleic acid RNA bridges DNA and protein synthesis

· Transcription = the synthesis of RNA using info in DNA

· 2 nucleic acids written n different forms of the same language, and info is transcribed/rewritten from DNA to RNA, where DNA serves as a template for assembling a complementary sequence of RNA

· Resulting RNA molecules = messenger RNA/mRNA because it carries a genetic message from DNA to protein synthesizing machinery

· Translation = synthesis of a polypeptide using info from mRNA

· Change in language: cell must translate nucleotide sequence of mRNA into amino acid sequence of a polypeptide

· Ribosomes = translation sites

· Transcription and translation occur in all organisms

· Basic mechanics for bacteria and eukaryotes are similar but difference in flow of genetic info

· Because bacteria don’t have nuclei their DNA is not separated by nuclear membranes from ribosomes

· Lack of compartmentalization allows translation of mRNA to begin while transcription is still in progress

· In a eukaryotic cell the nuclear envelope separates transcription from translation

· Transcription occurs in the nucleus resulting in pre-mRNA

· Initial RNA transcript = primary transcript

· RNA transcripts are modified to produce final mRNA

· mRNA is transported to cytoplasm where translation occurs

· Central dogma dubbed by Francis crick: DNA RNA Protein

The Genetic Code

· Codons: Triplets of Nucleotides

· Problem: getting from four nucleotide bases to 20 amino acids

· Triplets of nucleotide bases are the smallest units of uniform length that can code for all amino acids 43=64 possible code words = more than enough to specify all amino acids

· Triplet code = genetic instructions for a polypeptide chain written in the DNA as a series of non-overlapping, three nucleotide words

· Transcribed into a complementary series of non-overlapping, three nucleotide words of mRNA

· Gene determines the sequence of nucleotide bases along mRNA

· For each gene only one of the two DNA strands is transcribed = template strand provides the patter for mRNA

· mRNA molecules is complementary rather than identical to its DNA template because RNA nucleotides are assembled on the template according to base-pairing rules (with U instead of T and ribose instead of deoxyribose)

· RNA molecule is synthesized in an antiparallel direction to the template DNA strand

· Codons are written in the 5’3’ direction

· Complementary DNA strand has the same code as the RNA strand (except with T instead of U) = coding strand

· Because codons are nucleotide triplets the number of nucleotides making up a genetic message must be three times the number of amino acids in the protein product

· Cracking the Code

· Cracked the genetic code of life when experiments disclosed the amino acid translations of each RNA codons

· First codon deciphered by Marshall Nirenberg by synthesizing an artificial mRNA by linking identical RNA nucleotides with uracil as the base: UUU

· Added to a test-tube with amino acids, ribosomes, and other components required translated UUU into polypeptide with units of phenylalanine amino acid

· Used same techniques for AAA, CCC, GGG

· More elaborate techniques were required to code mixed triplets, all 64 codons were deciphered

· Three codons don’t code- they code for stop signals marking the end

· AUG both codes for methionine and functions as a start signal

· There is a redundancy in the genetic code but no ambiguity

· Multiple codons can code for the same amino acid but never specify any other amino acid

· Often codons that are synonyms vary only in third nucleotide

· Need the correct reading frame to extract the intended message

· Wrong chain will be made if nucleotides are read from left to right (5’3’)

· Evolution of the Genetic Code

· The genetic code is nearly universal shared by most organisms

· Codons code for the same amino acids in every organism

· Some exceptions where codons differ from standard ones in unicellular eukaryotes

· Evolutionary significance of code’s near universality = language must have been operating very early in the history of life, early enough so be present in the common ancestor of all present day organisms

17.2: Transcription is the DNA-directed synthesis of RNA

Molecular Components of Transcription

· RNA polymerase pries the two strands of DNA apart and joins together RNA nucleotides complementary to the DNA template strand, elongating the RNA polynucleotide

· Assembles in the 5’3’ direction

· Unlike DNA polymerase it can start the chain from scratch w/o primer

· Specific sequences of nucleotides along DNA mark where transcription of a gene begins and ends

· RNA polymerase attaches and initiates transcription at the promoter DNA sequence

· Terminator = sequence that signals end of transcription in bacteria

· Promoter is upstream of the terminator

· Stretch of DNA between promoter and terminator that is transcribed into RNA = transcription unit

· Bacteria have one type of RNA polymerase that synthesizes not only mRNA but other RNA as well like ribosomal RNA

· Eukaryotes have at least three types of RNA polymerase in their nuclei

· RNA polymerase II used for mRNA synthesis

· Others transcribe RNA molecules that aren’t translated into protein

Synthesis of an RNA Transcript

· RNA Polymerase Binding and Initiation of Transcription

· Promoter includes start point=nucleotide where RNA synthesis begins

· Extends several dozen nucleotide pairs upstream from start

· RNA polymerase binds in a precise location and orientation on the promoter determining where transcription starts and which of the two strands of the helix is used as the template

· Transcription factors = collection of proteins that mediate the binding of RNA polymerase and the initiation of transcription in eukaryotes

· Only after they are attached to promoter does RNA polymerase II bind transcription initiation complex

· TATA box = DNA sequence that helps it form

· Protein=protein interactions in controlling eukaryotic transcription

· Elongation of the RNA Strand

· As RNA polymerase moves along DNA it continues to untwist double helix, exposing 10-20 nucleotides at a time for pairing with RNA

· Adds nucleotides to the 3’ end of the growing RNA molecule

· The new RNA molecule peels away from its template and the DNA double helix reforms

· A single gene can be transcribed simultaneously by sever molecules of RNA polymerase following each other

· Growing strand of RNA trails off from each polymerase

· Congregation of many polymerase molecules simultaneously transcribing a single gene increases about of mRNA transcribed from it

· Termination of Transcription

· In bacteria: transcription proceeds through DNA terminator sequence

· Transcribed terminator functions as termination signal causing polymerase to detach from DNA and release transcript

· Requires no further modification

· In eukaryotes: RNA polymerase II transcribes DNA polyadenylation signal sequence which codes for signal in pre-mRNA (AAUAAA)

· At a point 10-35 nucleotides downstream from the signal proteins associated with RNA transcript cut it free from the polymerase releasing the pre-mRNA

17.3: Eukaryotic cells modify RNA after transcription

Eukaryotic cells modify RNA after transcription

· RNA processing = when enzymes in the eukaryotic nucleus modify pre-mRNA before its dispatched to the cytoplasm

· Alteration of mRNA Ends

· At the 5’ end receives a 5’ cap, a modified form of guanine added after the first 20-40 nucleotides

· At 3’ end enzyme adds 50-250 adenine nucleotides forming poly-A tail

· Cap and tail functions:

· Facilitate the export of mRNA form the nucleus

· Protect mRNA from degradation by hydrolytic enzymes

· Help ribosomes attach to the 5’ end in the cytoplasm

· Split Genes and RNA Splicing

· RNA splicing = removal of portions of RNA = cut and paste job

· Average transcription unit is 27,000 nucleotides but it only takes 1,200 nucleotides to code for the average protein

· Most genes and RNA transcripts have long noncoding stretches which are interspersed between coding segments

· Introns = noncoding segments/intervening sequences

· Exons = other regions that are expressed/translated

· Exit the nucleus

· RNA polymerase translates introns and exons but the introns are cut out and the exons joined together

· Short nucleotide sequence at each end of an intron gives the signal for RNA splicing

· Small nuclear ribonucleoproteins/snRNPs, located in the nucleus recognize splice sites

· snRNA = RNA in a snRNP

· Several snRNPs join other proteins to form spliceosome

· Interacts with sites along an intron, releasing the intron which is degraded and joining the exons

· Catalyze processes as well as participate in it

· Ribozymes = RNA molecules that function as enzymes

· In some organisms the intron RNA functions as a ribozyme and catalyzes its own excision = self-splicing

· RNA is single-stranded so a region can base-pair with a complementary region elsewhere on the same molecule giving it a 3-D structure

· Some of the bases in RNA contain functional groups that participate in catalysis

· The ability of RNA to hydrogen-bond with other nucleic acids adds specificity to catalytic activity complementary base paring between RNA of splicesome and RNA of primary RNA precisely locates the region to catalyze splicing

· The Functional and Evolutionary Importance of Introns: Evolution

· Specific functions have not been identified for most introns but some contain sequences that regulate gene expression effecting products

· A single gene can encode more than one kind of polypeptide

· many genes are known to give rise to 2+ different polypeptides depending on which segments are treated as exons = alternative RNA splicing

· Ex: sex differentiation in fruit flies

· Proteins have a modular architecture with discrete structural and functional regions = domains

· Different exons code for different domains of a protein

· Ex: include an active site

· Introns may facilitate evolution of new and potentially beneficial proteins through exon shuffling

· Introns increase the probability of crossing over between the exons of alleles of a gene by providing more terrain for crossovers without interrupting code sequences

· new combinations of exons and proteins with altered structure and function

· Occasional mixing and matching of exons between different genes

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

Molecular Components of Translation

· During translation a cell reads a genetic message and builds a polypeptide

· Translator = transfer RNA/tRNA used to transfer amino acids from the cytoplasmic pool of amino acids to a growing polypeptide in a ribosome

· A cell keeps its cytoplasm stocked will all 20 amino acids either by synthesizing them from other compounds or by taking them up from the surrounding solution

· The Structure and Function of Transfer RNA

· tRNA are not all identical, each type of tRNA molecule translates a particular mRNA codon into an amino acid

· tRNA arrives at a ribosome bearing a specific amino acid at one end

· At the other end it has an anticodon which base pairs with a complementary codon on mRNA

· As an mRNA molecule is moved through a ribosome the amino acid will be added to the polypeptide chain whenever the codon is presented for translation

· Codon by codon the genetic message is translated as tRNAs deposit amino acids and the ribosome joins the amino acids in the chain

· tRNA molecule is a translator between a codon to an amino acid

· tRNA is transcribed from DNA templates

· Each tRNA is used repeatedly

· tRNA consists of a single RNA strand that is ~80 nucleotides long

· Has complementary stretches of nucleotide bases that can hydrogen bond to each other the strand can fold back upon itself and form a molecule with a 3D structure

· Amino acid attaches at the 3’ end

· Translation requires two instances of molecular recognition:

· tRNA binds to an mRNA codon specifying a particular amino acid must carry that specific amino acid to the ribosome Aminoacyl-tRNA synthases carry out matching

· Active site of each type only fits a specific combination of amino acid and tRNA

· 20 different synthases, one for each amino acid

· Catalyzes the covalent attachment of the amino acid to its tRNA driven by hydrolysis of ATP aminoacyl tRNA (charged tRNA)

· Pairing of tRNA anticodon with the appropriate mRNA codon

· Some tRNAs bind to more than one codon because base pairing between the third nucleotide base of a codon and the corresponding base of the tRNA codon are relaxed compared to those at the other codon positions

· Wobble = flexible base pairing at third codon position

· Ribosomes facilitate the coupling of tRNA anticodons with mRNA codons

· Ribosomes are made up of a large and small subunit made of ribosomal RNAs (rRNAs) proteins

· In eukaryotes subunits are made in the nucleolus

· Ribosomal RNA genes are transcribe and RNA is processed and assembled with proteins imported from cytoplasm

· Large and small subunits join to form a functional ribosome when they attach to an mRNA molecule

· Most abundant type of cellular RNA

· Eukaryotic ribosomes are large and differ in molecular composition from bacterial ribosomes

· Some drugs can inactivate bacterial ribosomes without inactivating eukaryotic ribosomes

· Ribosome structure reflects its function of bringing together mRNA and tRNA with amino acids:

· 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 to be added to the chain

· E site (exit site) is where discharged tRNAs leave

· The ribosome holds the tRNA and mRNA in close proximity and positions the new amino acid for addition to the carboxyl end of the growing polypeptide

· Catalyzes formation of the peptide bond

· rRNA, not protein, is primarily responsible for both structure and function of the ribosme

· Proteins support the shape changes of the rRNA molecules as they carry out catalysis during translation

Building a Polypeptide

· Divide translation into three stages: initiation, elongation, and termination

· Energy is required, provided by hydrolysis of GTP (similar to ATP)

· Ribosome Association and Initiation of Translation

· Initiation stage of translation brings together mRNA, tRNA with the first amino acid of the polypeptide and the two subunits of a ribosome

· First a small ribosomal subunit binds to mRNA and initiator tRNA which carries methionine

· Complementary base pairing between site and rRNA involved

· In eukaryotes the small subunit (with initiator tRNA bound) binds to the 5’ cap of mRNA and then scans downstream along mRNA until it reaches the start codon

· Initiator tRNA hydrogen-bonds to AUG start codon, signaling start of translation and establishing the codon reading frame

· Next the large ribosomal subunit attaches completing the translation initiation complex

· At the end the initiator tRNA sits in the P site of the ribosome and the vacant A site is ready for the next tRNA

· Initiation factors are required to bring all the pieces together

· Cell expends energy through hydrolysis of GTP to form initiation complex

· Polypeptide is always synthesized in one direction, from the N-terminus end (methionine) toward C-terminus (the final amino acid)

· Elongation of the Polypeptide Chain

· Elongation stage adds amino acids one by one to the previous amino acid at the C-terminus end of the growing chain

· Each addition involves several elongation factor proteins

· mRNA is moved through ribosome in one direction, 5’3’ end

· Move relative to each other, codon by codon

· Occurs in three step cycle (energy used in steps 1 and 3), see picture

1. Codon recognition: anticodon of incoming tRNA base pairs with complementary mRNA codon in the A site

a. Hydrolysis of GTP increases accuracy and efficiency

2. Peptide bond formation: rRNA molecule of the large ribosomal subunit catalyzes the formation of a peptide bond between amino group of the new amino acid at the A site and the carboxyl end of the growing chain in the P site

a. Removes polypeptide form tRNA in the P site and attaches it to the amino acid on the tRNA in the A site

3. Translocation: ribosome translocates tRNA in A site to the P site

a. Empty tRNA in the P site is moved to the E site where it’s released

b. mRNA moves along with its bound tRNAs, bringing the next codon to be translated into the A site

· Termination of Translation

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

· Release factor = protein shaped like tRNA binds directly to the stop codon in the A site, causing the addition of a water molecule instead of an amino acid to the polypeptide chain

· breaks (hydrolyzes) the bond between the completed polypeptide and the tRNA in the P site, releasing the polypeptide through the E site

· Translation assembly comes apart

· Polyribosomes:

· A single ribosome can make a polypeptide in less than a minute

· Multiple ribosomes translate an mRNA at the same time- a single mRNA is used to make many copies of a polypeptide simultaneously

· Once a ribosome is far enough past the start codon a second ribosome can attach to the mRNA, resulting in a number of ribosomes trailing along the mRNA = polyribosomes/polysomes

· Allow a cell to make many copies of polypeptide very quickly

Completing and Targeting the Functional Protein

· Protein Folding and Post-Translational Modifications

· Translation isn’t enough to make a functional protein

· During synthesis the polypeptide chain begins to coil and fold spontaneously due to its amino acid sequence (primary structure)

· Chaperonin protein helps the polypeptide fold correctly

· Post-translational modifications may be required before the protein can begin doing its job

· Amino acids may be chemically modified by attachment of sugars, lipids, phosphate groups, etc.

· Enzymes may remove one or more amino acids from les the leading end or enzymatically cleave the chain

· Two or more polypeptides are synthesized separately and may come together becoming a protein of quaternary structure

· Targeting Polypeptides to Specific Locations

· Two types of ribosomes are found in the cell: free and bound

· Free are suspended in cytosol

· Synthesize proteins that stay in the cytosol

· Bound are attached to the endoplasmic reticulum or the nuclear envelope

· Make proteins of the endomembrane system and proteins secreted from the cell

· Polypeptide synthesis always begins in the cytosol as a free ribosome starts to translate mRNA

· Continues to completion unless the growing polypeptide cues the ribosome to attach to the ER

· Polypeptides destined for the endomembrane system or secretion are marked by a signal peptide which targets the protein to the ER

· Sequence of 20 amino acids near the end

· Recognized as it emerges from the ribosome by signal-recognition particle (SRP) protein-RNA complex

· Functions as an escort bringing the ribosome to a receptor protein on the ER membrane

· Part of a multiprotein translocation complex where synthesis continues, the growing polypeptide snaking across the membrane into ER lumen via protein pore

· Removed by an enzyme and either is released or remains embedded

· Other types are used to target polypeptides to mitochondria, chloroplasts, interior of the nucleus, and other organelles not part of the endomembrane system

· Translation is completed in the cytosol before the polypeptide is imported into the organelle

· Bacteria use to target proteins to membrane for secretion

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

Types of Small-Scale Mutations

· Mutations = changes in genetic info of a cell, responsible for diversity of genes in organisms (source of new genes)

· Point mutations = changes in a single nucleotide pair of a gene

· If it occurs in a gamete that gives rise to gametes it can be transmitted to offspring and future generations

· Ex: hemoglobin: single point mutation that encodes polypeptide of hemoglobin leading to production of abnormal protein

· Nucleotide-pair substitution = replacement of one nucleotide and its partner with another pair of nucleotides

· Some have no phenotypic effect because of redundancy of the genetic code = silent mutation

· Missense mutations do change the amino acid can or can’t have a phenotypic effect

· Nonsense mutation = missense mutation that changes a codon to a stop codon terminating translation prematurely

· nonfunctional proteins

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

· Have a disastrous effect because they alter the reading frame = frameshift mutation all nucleotides that are downstream of the deletion/insertion will be improperly grouped into codons

· nonfunctional proteins

Mutagens = physical and chemical agents that interact with DNA causing mutations

· Incorrect base will be used as a template in the next round of replication = spontaneous mutations

· Change is passed on to the next generation of cells

· Hermann Muller discovered that X-rays caused genetic changes in fruit flies

· Pose hazards to genetic material of people

· Chemical mutagens fall into many categories

· Nucleotide analogs = chemicals similar to normal DNA nucleotides but pair incorrectly during replication

· Others interfere with correct DNA replication by inserting themselves into the DNA, distorting the double helix

· Others cause chemical changes in bases changing pairing properties

· Tests of mutagenic activity of chemicals used to identify carcinogens

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

Comparing Gene Expression in Bacteria, Archaea, and Eukarya

· Bacterial and eukaryotic RNA polymerases differ (archaeal RNA polymerase resembles eukaryotic ones)

· Archaea and eukaryotes use transcription factors, unlike accessory proteins in bacteria

· Transcription is terminated differently between eukaryote and bacteria (Achaea similar to eukaryotes probs)

· Archaeal ribosomes are the same size as bacterial ribosomes but sensitivities to chemical inhibitors are more similar to eukaryotic ribosomes

· Initiation of translation is different in bacteria and eukaryotes (archaea is more like bacteria)

· Most important differences between bacteria and eukaryotes arise from bacteria’s lack of compartmental organization

· Streamlined operation in a bacterial cell- without a nucleus it can simultaneously transcribe and translate the same gene

· Newly made protein quickly diffuses to site of function

· Similar in archaeal cells which lack a nuclear envelope

· Eukaryotic cell’s nuclear envelope segregates transcription from translation providing a compartment for RNA processing

· Includes additional steps to coordinate elaborate activities

What Is a Gene? Revisiting the Question

· Functional definition of a gene as a DNA sequence that codes for a specific polypeptide chain

· Most eukaryotic genes contain noncoding segments so large portions of these genes have no corresponding segments in polypeptides

· Promoters and regulatory regions considered part of the gene

· DNA that transcribes rRNA, tRNA, and other RNAs that aren’t translated are also part of the gene

· A gene is a region of DNA that can be expressed to produce a final functional product that is either a polypeptide or an RNA molecule