Basic Principles of Transcription and Translation

Transcription

1 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 The ribosome is part of the cellular machinery for translation, polypeptide synthesis Basic Principles of Transcription and Translation RNA is the intermediate between genes and the proteins for which they code Transcription is the synthesis of RNA under the direction of DNA Transcription produces messenger RNA (mrna) Translation is the synthesis of a polypeptide, which occurs under the direction of mrna Ribosomes are the sites of translation

2 In prokaryotes, mrna produced by transcription is immediately translated without more processing In a eukaryotic cell, the nuclear envelope separates transcription from translation Eukaryotic RNA transcripts are modified through RNA processing to yield finished mrna A primary transcript is the initial RNA transcript from any gene The central dogma is the concept that cells are governed by a cellular chain of command: DNA RNA protein Eukaryotic RNA transcripts are modified through RNA processing to yield finished mrna TRANSCRIPTION TRANSLATION Polypeptide DNA mrna Ribosome a) Bacterial cell. In a bacterial cell which lacks a nucleus, mrna produced by transcription is immediately translated without additional processing. (a) Bacterial cell TRANSCRIPTION DNA Nuclear envelope b) Eukaryotic cell. The nucleus provides a separate compartment for transcription. The original RNA transcript called pre mrna is processed in various ways before leaving the nucleus as mrna. RNA PROCESSING Pre-mRNA mrna TRANSLATION Ribosome Polypeptide (b) Eukaryotic cell Overview: the roles of transcription and translation in the flow of genetic information. In a cell inherited information flows from DNA to RNA to protein. The two main stages of information flow are transcription and translation.

3 TRANSCRIPTION DNA mrna (a) Bacterial cell TRANSCRIPTION TRANSLATION Polypeptide DNA mrna Ribosome Nuclear envelope TRANSCRIPTION DNA Pre-mRNA (b) Eukaryotic cell

4 Nuclear envelope TRANSCRIPTION DNA RNA PROCESSING Pre-mRNA mrna (b) Eukaryotic cell Nuclear envelope TRANSCRIPTION DNA RNA PROCESSING Pre-mRNA mrna TRANSLATION Ribosome Polypeptide (b) Eukaryotic cell

5 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 bases correspond to an amino acid? Codons: Triplets of Bases The flow of information from gene to protein is based on a triplet code: a series of nonoverlapping, three-nucleotide words These triplets are the smallest units of uniform length that can code for all the amino acids Example: AGT at a particular position on a DNA strand results in the placement of the amino acid serine at the corresponding position of the polypeptide to be produced

6 During transcription, one of the two DNA strands called the template strand provides a template for ordering the sequence of nucleotides in an RNA transcript During translation, the mrna base triplets, called codons, are read in the to direction Each codon specifies the amino acid to be placed at the corresponding position along a polypeptide Colons along an mrna molecule are read by translation machinery in the to direction Each codon specifies the addition of one of 20 amino acids DNA molecule Gene 1 Gene 2 DNA template strand TRANSCRIPTION mrna Codon TRANSLATION Protein Amino acid Gene 3 The triplet code. For each gene, one strand of DNA functions as a template for transcription. The base pairing rules for DNA synthesis also guide transcription, but uracil (U) takes the place of thymine (T) in RNA. During translation the mrna is read as a sequence of base triplets called codons. Each codon specifies an amino acid to be added to the growing polypeptide chain. The mrna is read in the 5 3 direction.

7 Cracking the Code All 64 codons were deciphered by the mid-1960s Of the 64 triplets, 61 code for amino acids; 3 triplets are stop signals to end translation 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 Second mrna base First mrna base ( end of codon) Third mrna base ( end of codon) The dictionary of the genetic code. The three bases of an mrna codon are designated here as the first, second and third bases reading in the 5 3 direction along the mrna. The codon AUG not only stands for the amino acid methionine (Met) but also functions as a start signal for ribosomes to begin translating the mrna at that point. Three of the 64 codons function as stop signals marking the end of a genetic message

8 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 Because diverse forms of life share a common genetic code, one species can be programmed to produce proteins characteristic of a second species by introducing DNA from the second species into the first Transcription is the DNA-directed synthesis of RNA: a closer look Transcription, the first stage of gene expression, can be examined in more detail The three stages of transcription: Initiation Elongation Termination

9 Molecular Components of Transcription RNA synthesis is catalyzed by RNA polymerase, which 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 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 Promoter Transcription unit DNA Start point RNA polymerase 1 Initiation Unwound DNA RNA transcript Template strand of DNA 2 Elongation Elongation Nontemplate strand of DNA RNA polymerase end RNA nucleotides Rewound DNA RNA transcript 3 Termination Completed RNA transcript Newly made RNA Direction of transcription ( downstream ) Template strand of DNA

10 Promoter Start point RNA polymerase Unwound DNA Rewound DNA RNA transcript Transcription unit RNA transcript DNA 1 Initiation Template strand of DNA 2 Elongation 3 Termination Completed RNA transcript 1) Initiation. After RNA polymerase binds to the promoter, the DNA strands unwind and the polymerase initiates RNA synthesis at the start point on the template strand. 2) 2) Elongation The polymerase moves downstream unwinding the DNA and elongating the RNA transcript 5 3 In the wake of transcription the DNA strands re-form a double helix. 3) 3) Termination Eventually the RNA transcript is released and the polymerase detaches from the DNA The stages of transcription: initiation elongation and termination. Thus general depiction of transcription applies to both bacteria and eukaryotes but the details of termination differ, as described in the text. Also in a bacterium the transcript is immediately usable as mrna in a eukaryote the RNA transcript must first undergo processing. Elongation Nontemplate strand of DNA RNA polymerase RNA nucleotides end Newly made RNA Direction of transcription ( downstream ) Template strand of DNA

11 RNA Polymerase Binding and Initiation of Transcription Promoters signal the initiation of RNA synthesis 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 1 Promoter Template TATA box Start point Template DNA strand Transcription factors RNA polymerase II 2 3 Transcription factors RNA transcript Transcription initiation complex 1) A eukaryotic promoter commonly includes a TATA box a nucleotide sequence containing TATA about 25 nucleotides upstream from the transcription start point. By convention nucleotide sequences are given as they occur on the nontemplate strand 2) Several transcription factors one recognizing the TATA box must bind to the DNA before RNA polymerase II can do so. 3) Additional transcription factors (purple) bind to the DNA along with RNA polymerase II forming the transcription initiation complex. The DNA double helix then unwinds and RNA synthesis begins at the start point on the template strand. The initiation of transcription at a eukaryotic promoter. In eukaryotic cells proteins called transcription factors mediate the initiation of transcription by RNA polymerase II

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

13 Eukaryotic cells modify RNA after transcription Enzymes in the eukaryotic nucleus modify pre-mrna 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 end receives a modified nucleotide cap The end gets a poly-a tail These modifications share several functions: They seem to facilitate the export of mrna They protect mrna from hydrolytic enzymes They help ribosomes attach to the end

14 Protein-coding segment Polyadenylation signal G P P P AAUAAA AAA AAA Cap UTR Start codon Stop codon UTR Poly-A tail RNA processing addition of the 5 cap and poly A tail. Enzymes modify the two ends of a eukaryotic pre mrna molecule. The modified ends may promote the export of mrna from the nucleus and they help protect the mrna from degradation. When the mrna reaches the cytoplasm the modified ends in conjunction with certain cytoplasmic proteins facilitate ribosome attachment. The 5 cap and poly A tail are not translated into protein, nor are the regions called the 5 untranslated regions (5 UTR) and 3 untranslated regions (3 UTR) 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

15 Exon Intron Exon Intron Pre-mRNA Cap Exon Poly-A tail 146 Coding segment Introns cut out and exons spliced together mrna Cap Poly-A tail UTR UTR RNA processing: mrna splicing. The RNA molecule shown here codes for β globin one of the polypeptides of hemoglobin. The numbers under the RNA refer to the codons. β globin is 146 amino acids long. The β globin gene and its pre mrna transcript have three exons corresponding to sequences that will leave the nucleus as RNA. (The 5 UTR and 3 UTR are parts of exons because they are included in the mrna however they do not code for protein). During RNA processing the introns are cut out and the exons are spliced together. In many genes the introns are much larger relative to the exons than they are in the β globin gene. The mrna is not drawn to scale. 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

16 Protein snrna RNA transcript (pre-mrna) Exon 1 Intron Exon 2 snrnps Spliceosome components Spliceosome mrna Exon 1 Exon 2 Other proteins Cut-out intron The roles of snrnps and spliceosomes in pre mrna splicing. The diagram shows only a portion of the pre mrna transcript, additional introns and exons lie downstream from the pictured ones here. 1) Small nuclear ribonucleoprotein (snrnps) and other proteins form a molecular complex called a spliceosome on a pre mrna molecules containing exons and introns. 2) Within the spliceosome snrna base pairs with nucleotides at specific sites along the intron. 3) The spliceosome cuts the pre mrna releasing the intron and at the same time splices the exons together. The spliceosome then comes apart releasing mrna which now contains only exons. 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 RNA may hydrogen-bond with other nucleic acid molecules

17 The Functional and Evolutionary Importance of Introns Some genes can encode more than one kind of polypeptide, depending on which segments are treated as exons during RNA splicing Such variations are called alternative RNA splicing Because of alternative splicing, 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

18 DNA Gene Exon 1 Intron Exon 2 Intron Exon 3 Transcription RNA processing Translation Correspondence between exons and protein domains. Domain 3 Domain 2 Domain 1 Polypeptide Translation is the RNA-directed synthesis of a polypeptide: a closer look A cell translates an mrna message into protein with the help of transfer RNA (trna) 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

19 Polypeptide Trp Phe Ribosome Amino acids trna with amino acid attached Gly trna Anticodon Translation: the basic concept. As a molecule of mrna is moved through a ribosome codons are translated into amino acids one by one. The interpreters are trna molecules, each type with a specific anticodon at one end and a corresponding amino acid at the other end. A trna adds its amino acid cargo to a growing polypeptide chain when the anticodon hydrogen bonds to a complementary codon on the mrna. Codons mrna The Structure and Function of Transfer RNA A trna molecule consists of a single RNA strand that is only about 80 nucleotides long A C C 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

20 Amino acid attachment site Anticodon (a) Two-dimensional structure Anticodon Hydrogen bonds Hydrogen bonds (b) Three-dimensional structure Amino acid attachment site Anticodon (c) Symbol used in this book Two dimensional structure. The four base paired regions and three loops are characteristic of all trnas as is the base sequence of the amino acid attachment site at the 3 end. The anticodon triplet is unique to each trna type as are some sequences in the other two loops. (the asterisks mark bases that have been chemically modified a characteristic of trna) The structure of transfer RNA (trna). Anticodons are conventionally written 3 5 to align properly with codons written 5 3. For base pairing RNA strands must be antiparallel like DNA. For example anticodon 3 AAG 5 pairs with mrna codon 5 UUC 3 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

21 Amino acid P P P ATP Adenosine Aminoacyl-tRNA synthetase (enzyme) 1) Active site binds to the amino acid and ATP 2) ATP loses two P groups and joins amino acids as AMP trna P Adenosine P P i P i P i trna Aminoacyl-tRNA synthetase 3) Appropriate trna covalently bonds to amino acid displacing AMP 4) The trna charged with amino acid is released by the enzyme P Adenosine AMP Aminoacyl-tRNA ( charged trna ) Computer model An aminoacyl trna synthethase joining a specific amino acid to a trna. Linkage of the trna and amino acid is an endergonic process that occurs at the expense of ATP. The ATP loses two phosphate groups becoming AMP (adenosine monophosphate) 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)

22 trna molecules Growing polypeptide E P A Exit tunnel Large subunit Small subunit mrna (a) Computer model of functioning ribosome P site (Peptidyl-tRNA binding site) E site (Exit site) mrna binding site E P A A site (AminoacyltRNA binding site) Large subunit Small subunit (b) Schematic model showing binding sites Amino end mrna E Codons Growing polypeptide Next amino acid to be added to polypeptide chain trna (c) Schematic model with mrna and trna a) Computer model of functioning ribosome. This is a model of a bacterial ribosome showing its overall shape. The eukaryotic ribosome is roughly similar. A ribosomal subunit is an aggregate of ribosomal RNA molecules and proteins b) Schematic model showing binding sites. A ribosome has an mrna binding site and three trna binding sites known as the A, P and E sites. c) Schematic model with mrna and trna. A trna fits into a binding site when its anticodon base pairs with an mrna codon. The P site holds the trna attached to the growing polypetide. The A site holds the trna carrying the next amino acid to be added to the polypeptide chain. Discharged trnas leaves from the E site. The anatomy of a functioning ribosome. 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

23 Building a Polypeptide The three stages of translation: Initiation Elongation Termination All three stages require protein factors 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

24 Met U A C A U G P site Met Large ribosomal subunit Initiator trna mrna Start codon mrna binding site GTP Small ribosomal subunit GDP E A Translation initiation complex 1) A small ribosomal subunit binds to a molecule of mrna. In a bacterial cell the mrna binding site on this subunit recognizes a specific nucleotide sequence on the mrna just upstream of the start codon. An initiator trna with the anticodon UAC base pairs with the start codon, AUG. This trna carries the amino acid methionine Met) 2) The arrival of a large ribosomal subunit completes the initiation complex. Proteins called initiation factors (not shown) are required to bring all the translation components together GTP provides the energy for the assembly. The initiator trna is in the P site, the A site is available to the trna bearing the next amino acid. The initiation of translation Elongation of the Polypeptide Chain During the elongation stage, 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 bond formation, and translocation

25 The elongation cycle of translation. The hydrolysis of GTP plays an important role in the elongation process Ribosome ready for next aminoacyl trna Amino end of polypeptide mrna E P site site A GTP 1) Codon recognition. The anticodon of an incoming aminoacyl trna base pairs with the complementary mrna codon in the A site. Hydrolysis of GTP increases the accuracy and efficiency of this step GDP E E P A P A 3) Translocation The ribosome translocates the trna in the A to the P site. The empty trna in the P site is moved to the E site where it is released. The mrna moves along with its bound trnas bringing the next codon to be translated into the A site GDP GTP E P A 2) Peptide bond formation. An rrna molecule of the large ribosomal subunit catalyses the formation of a peptide bond between the new amino acid in the A site and the carboxyl end of the growing polypeptide in the P site. This step removes the polypeptide from the trna in the P site and attaches it to the amino acid on the trna in the A site 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

26 Release factor Free polypeptide Stop codon (UAG, UAA, or UGA) When a ribosome reaches a stop codon on mrna, the A site of the ribosome accepts a release factor a protein shaped like a trna instead of an aminoacyl trna, The release factor hydrolyzes the bond between the trna in the P site and the last amino acid of the polypeptide chain. The polypeptide is thus freed from the ribosome. The two ribosomal subunits and the other components of the assembly dissociate. The termination of translation. Like elongation, termination requires GTP hydrolysis as well as additional factors which are not shown here 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

27 Growing polypeptides Completed polypeptides Incoming ribosomal subunits Start of mrna ( end) Polyribosome End of mrna ( end) An mrna molecule is generally translated simultaneously by several ribosomes in clusters called polyribosomes. Ribosomes mrna 0.1 µm This micrograph shows a large polyribosome in a prokaryotic cell (TEM). 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

28 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

29 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 1) Polypetide synthesis begins on a free ribosomo in the cytosol. 2) An SRP binds to the signal peptide. halting synthesis momentarily 3) The SRP binds to a receptor protein in the ER membrane. This receptor is part of a protein complex (a translocation complex) that has a membrane pore and a signal cleaving enzyme 4) The SRP leaves and 5) The signal polypetides synthesis cleaving resumes with enzyme simultaneous cuts off the translocation across the signal membrane (The signal peptide peptide stays attached to the translocation complex) 6) The rest of the completed polypeptide leaves the ribosome and folds into its final conformation Ribosome Signal peptide Signalrecognition particle (SRP) CYTOSOL ER LUMEN mrna Translocation complex SRP receptor protein Signal peptide removed ER membrane Protein The signal mechanism for targeting proteins to the ER. A polypeptide destined for the endomembrane system or for secretion from the cell begins with a signal peptide a series of amino acids that targets it for the ER. This figure shows the synthesis of a secretory protein and its simultaneous import into the ER. In the ER and then in the Golgi, the protein will be processed further. Finally a transport vesicle will convey it to the plasma membrane for release from the cell

30 RNA plays multiple roles in the cell: a review Type of RNA Messenger RNA (mrna) Transfer RNA (trna) Ribosomal RNA (rrna) Functions Carries information specifying amino acid sequences of proteins from DNA to ribosomes Serves as adapter molecule in protein synthesis; translates mrna codons into amino acids Plays catalytic (ribozyme) roles and structural roles in ribosomes Type of RNA Primary transcript Small nuclear RNA (snrna) SRP RNA Functions Serves as a precursor to mrna, rrna, or trna, before being processed by splicing or cleavage Plays structural and catalytic roles in spliceosomes Is a component of the the signalrecognition particle (SRP)

31 Type of RNA Small nucleolar RNA (snorna) Small interfering RNA (sirna) and microrna (mirna) Functions Aids in processing pre-rrna transcripts for ribosome subunit formation in the nucleolus Are involved in regulation of gene expression RNA s diverse functions range from structural to informational to catalytic Properties that enable RNA to perform many different functions: Can hydrogen-bond to other nucleic acids Can assume a three-dimensional shape Has functional groups that allow it to act as a catalyst (ribozyme)

32 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 addition extensive RNA processing occurs in the nucleus In archaea, transcription and translation are likely coupled

33 RNA polymerase RNA polymerase Polyribosome Polypeptide (amino end) DNA Polyribosome Direction of transcription Ribosome mrna ( end) mrna 0.25 µm DNA Coupled transcription and translation in bacteria. In bacterial cells, the translation of mrna can begin as soon as the leading (5 ) end of the mrna molecule peels away from the DNA template. The micrograph (TEM) shows a strand of E coli DNA being transcribed by RNA polymerase molecules. Attached to each RNA polymerase molecule is a growing strand of mrna which is already being translated by ribosomes. The newly synthesized polypeptides are not visible in the micrograph but are shown in the diagram. What Is a Gene? Revisiting the Question The idea of the gene itself is a unifying concept of life 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

34 A summary of transcription and translation in a eukaryotic cell 1) Transcription- RNA is transcribed from a DNA template. 2) RNA processing- In eukaryotes the RNA transcript (pre mrna) is spliced and modified to produce mrna, which moves from the nucleus to the cytoplasm 3) The mrna leaves the nucleus and attaches to a ribosome 4) Amino acid activation- Each amino acid attaches to its proper trna with the help of a specific enzyme and ATP. 5) Translation- A succession of trnas add their amino acids to the polypeptide chain as the mrna is moved through the ribosome one codon at a time (When completed the polypeptide is released from the ribosome Conducting the Genetic Orchestra Prokaryotes and eukaryotes alter gene expression in response to their changing environment In multicellular eukaryotes, gene expression regulates development and is responsible for differences in cell types RNA molecules play many roles in regulating gene expression in eukaryotes

35 Individual bacteria respond to environmental change by regulating their gene expression A bacterium can tune its metabolism to the changing environment and food sources This metabolic control occurs on two levels: Adjusting activity of metabolic enzymes Regulating genes that encode metabolic enzymes Bacteria often respond to environmental change by regulating transcription Natural selection has favored bacteria that produce only the products needed by that cell A cell can regulate the production of enzymes by feedback inhibition or by gene regulation Gene expression in bacteria is controlled by the operon model

36 Operons: The Basic Concept An operon is the entire stretch of DNA that includes the operator, the promoter, and the genes that they control In bacteria, genes are often clustered into operons, composed of An operator, an on-off switch A promoter Genes for metabolic enzymes A cluster of functionally related genes can be under coordinated control by a single on-off switch The regulatory switch is a segment of DNA called an operator usually positioned within the promoter The operon can be switched off by a protein repressor The repressor prevents gene transcription by binding to the operator and blocking RNA polymerase The repressor is the product of a separate regulatory gene The repressor can be in an active or inactive form, depending on the presence of other molecules A corepressor is a molecule that cooperates with a repressor protein to switch an operon off

37 Eukaryotic gene expression can be regulated at any stage All organisms must regulate which genes are expressed at any given time In multicellular organisms gene expression is essential for cell specialization Differential Gene Expression Almost all the cells in an organism are genetically identical Differences between cell types result from differential gene expression, the expression of different genes by cells with the same genome In each type of differentiated cell, a unique subset of genes is expressed Many key stages of gene expression can be regulated in eukaryotic cells Errors in gene expression can lead to diseases including cancer Gene expression is regulated at many stages

38 All our cells start off with the same set of genes A small percentage of these genes are expressed in all our cells- housekeeping genes like for example for glycolysis Through development and our lives different cells selectively express different genes For example RBCs transcribe hemoglobin genes whereas the eye does not transcribe this gene and instead it expresses crystalline Signal DNA RNA Cap Gene NUCLEUS Chromatin Chromatin modification DNA unpacking involving histone acetylation and DNA demethylation Exon Intron Tail Gene available for transcription Transcription Primary transcript RNA processing mrna in nucleus Transport to cytoplasm CYTOPLASM Stages in gene expression that can be regulated in eukaryotic cells. In this diagram the colored boxes indicate the processes most often regulated; each color indicates the type pf molecule that is affected (blue=dna, orange= RNA, purple= protein). The nuclear envelope separating transcription from translation in eukaryotic cells offers an opportunity for post transcriptional control in the form of RNA processing that is absent in prokaryotes. In addition eukaryotes have a greater variety of control mechanisms operating before transcription and after translation. The expression of any given gene, however does not necessarily involve every stage shown; for example not every polypeptide is cleaved.

39 CYTOPLASM mrna in cytoplasm Degradation of mrna Translation Polypeptide Protein processing such as cleavage and chemical modification Degradation of protein Active protein Transport to cellular destination Cellular function (such as enzymatic activity, structural support etc.) Controls before transcription Promoters Enhancers Methylation and acetylation Rearrangement- multiplication- for example polytene chromosomes contain several copies of genes allowing more RNA and subsequently more proteins to get produced

40 Regulation of Chromatin Structure Genes within highly packed heterochromatin are usually not expressed Chemical modifications to histones and DNA of chromatin influence both chromatin structure and gene expression Histone Modifications In histone acetylation, acetyl groups are attached to positively charged lysines in histone tails This process loosens chromatin structure, thereby promoting the initiation of transcription The addition of methyl groups (methylation) can condense chromatin; the addition of phosphate groups (phosphorylation) next to a methylated amino acid can loosen chromatin The histone code hypothesis proposes that specific combinations of modifications help determine chromatin configuration and influence transcription

41 Histone tails DNA double helix Amino acids available for chemical modification (a) Histone tails protrude outward from a nucleosome. This is an end view of a nucleosome. The amino acids in the Ntermincal tails are accessible for chemical modification Unacetylated histones Acetylated histones A simple model of histone tails and the effect of histone acetylation. In addition to acetylation histones can undergo several other types of modifications that also help determine the chromatin configuration in a region. (b) Acetylation of histone tails promotes loose chromatin structure that permits transcription. A region of chromatin in which nucleosomes are unacetylated forms a compact structure (left) in which the DNA is not transcribed. When nucleosomes are highly acetylated (right) the chromatin becomes less compact, and the DNA is accessible for transcription DNA Methylation DNA methylation, the addition of methyl groups to certain bases in DNA, is associated with reduced transcription in some species DNA methylation can cause long-term inactivation of genes in cellular differentiation In genomic imprinting, methylation regulates expression of either the maternal or paternal alleles of certain genes at the start of development

42 Chemical Modifications Methylation of DNA can inactivate genes Acetylation of histones allows DNA unpacking and transcription Methylation of histone or of DNA usually turns a gene off. Acetylation of histone usually turns a gene on.

43 Epigenetic Inheritance Although the chromatin modifications just discussed do not alter DNA sequence, they may be passed to future generations of cells The inheritance of traits transmitted by mechanisms not directly involving the nucleotide sequence is called epigenetic inheritance Regulation of Transcription Initiation Chromatin-modifying enzymes provide initial control of gene expression by making a region of DNA either more or less able to bind the transcription machinery

44 Organization of a Typical Eukaryotic Gene Associated with most eukaryotic genes are control elements, segments of noncoding DNA that help regulate transcription by binding certain proteins Control elements and the proteins they bind are critical to the precise regulation of gene expression in different cell types Enhancer (distal control elements) DNA Upstream Proximal control elements Primary RNA Transcript (pre-mrna) mrna Promoter Intron RNA Cap Exon Intron Exon Exon Intron Exon Intron Exon 5 UTR untranslated region Coding segment Start codon Poly-A signal sequence Termination region Intron Exon Transcription RNA processing Cap and tail added introns excised and exons spliced together Downstream Cleaved end of primary transcript Poly-A signal Stop codon UTR Poly-A tail A eukaryotic gene and its transcript. Each eukaryotic gene has a promoter a DNA sequence where RNA polymerase binds and starts transcription proceeding downstream. A number of control elements (gold) are involved in regulating the initiation of transcription; these are DNA sequences located near (proximal to) or far from (distal to) the promoter. Distal control elements can be grouped together as enhancers one of which is shown for this gene. A polyadenylation (poly-a) signal sequence in the last exon of the gene is transcribed into an RNA sequence that signals where the transcript is cleaved and the poly A tail added. Transcription may continue for hundreds of nucleotides beyond the poly A signal before terminating. RNA processing of the primary transcript into a functional mrna involves three steps: addition of the 5 cap addition of the poly A tail and splicing. In the cell the 5 cap is added soon after transcription is initiated splicing and poly A tail addition may also occur while transcription is till under way.

45 The Roles of Transcription Factors To initiate transcription, eukaryotic RNA polymerase requires the assistance of proteins called transcription factors General transcription factors are essential for the transcription of all protein-coding genes In eukaryotes, high levels of transcription of particular genes depend on control elements interacting with specific transcription factors Enhancers and Specific Transcription Factors Proximal control elements are located close to the promoter Distal control elements, groups of which are called enhancers, may be far away from a gene or even located in an intron An activator is a protein that binds to an enhancer and stimulates transcription of a gene Bound activators cause mediator proteins to interact with proteins at the promoter

46 DNA Enhancer Activators 1) Activator proteins bind to distal control elements grouped as an enhancer in the DNA. This enhancer has three binding sites. 2) A DNA bending protein brings the bound activators closer to the promoter. General transcription factors mediator proteins and RNA polymerase are nearby. 3) The activators bind to certain mediator proteins and general transcription factors, helping them form an active transcription initiation complex on the promoter. Distal control element DNA-bending protein Promoter TATA box General transcription factors Gene Group of mediator proteins A model for the action of enhancers and transcription activators: Bending of the DNA by a proteins enables enhancers the influence a promoter hundreds or even thousands of nucleotides away. Specific transcription factors called activators bind to the enhancer DNA sequences and then to a group of mediator proteins, which in turn bind to general transcription factors assembling the transcription initiation complex. These protein protein interactions facilitate the correct positioning of the complex on the promoter and the initiation of RNA synthesis. Only one enhancer (with three orange control elements) is shown here, but a gene may have several enhancers that act at different times or on different cell types Transcription initiation complex RNA polymerase II RNA polymerase II RNA synthesis Coordinately Controlled Genes in Eukaryotes Unlike the genes of a prokaryotic operon, each of the coordinately controlled eukaryotic genes has a promoter and control elements These genes can be scattered over different chromosomes, but each has the same combination of control elements Copies of the activators recognize specific control elements and promote simultaneous transcription of the genes

47 Mechanisms of Post-Transcriptional Regulation Transcription alone does not account for gene expression Regulatory mechanisms can operate at various stages after transcription Such mechanisms allow a cell to fine-tune gene expression rapidly in response to environmental changes Control of RNA processing In alternative RNA splicing, different mrna molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons and which as introns Alternative splicing- For example different muscle cells express slightly different forms of the troponin gene by utilizing alternative splicing. This generates proteins with somewhat unique functions Nuclear envelope- UTRs contain zipcodes which allow the RNA to exit the nucleus with the aid of proteins which recognize it and selectively bind to it. In addition these unique zipcodes specify to which cytoplasmic location these RNA must move. This is crucial during embryonic development.

48 Exons DNA Troponin T gene Primary RNA transcript mrna RNA splicing Alternative RNA splicing of the troponin T gene. The primary transcript of this gene can be spliced in more than one way, generating different mrna molecules. Notice that one mrna molecule has ended up with exon 3 (green) and the other with exon 4 (purple). These two mrnas are translated into different but related muscle proteins. or mrna Degradation The life span of mrna molecules in the cytoplasm is a key to determining protein synthesis Eukaryotic mrna is more long lived than prokaryotic mrna The mrna life span is determined in part by sequences in the leader and trailer regions

49 Initiation of Translation The initiation of translation of selected mrnas can be blocked by regulatory proteins that bind to sequences or structures of the mrna Alternatively, translation of all mrnas in a cell may be regulated simultaneously For example, translation initiation factors are simultaneously activated in an egg following fertilization Protein Processing and Degradation After translation, various types of protein processing, including cleavage and the addition of chemical groups, are subject to control Proteasomes are giant protein complexes that bind protein molecules and degrade them

50 1) Multiple ubiquitin molecules are attached to a protein by enzymes in the cytosol Ubiquitin 2) The ubiquitin tagged protein is recognized by a proteasome which unfolds the protein and sequesters it within a central cavity Proteasome 3) Enzymatic components of the proteasome cut the protein into small peptides which can be further degraded by other enzymes in the cytosol. Proteasome and ubiquitin to be recycled Protein to be degraded Ubiquitinated protein Protein entering a proteasome Protein fragments (peptides) Degradation of a protein by a proteasome. A proteasome, an enormous protein complex shaped like a trash can, chops up unneeded proteins in the cell. In most cases the proteins attacked by a proteasome have been tagged with short chains of ubquitin a small protein. Steps 1 and 3 require ATP. Eukaryotic proteasomes are as massive as ribosomal subunits and are distributed throughout the cell. Their shape somewhat resembles that of chaperone proteins, which protect protein structure rather than destroy it Control after translation (post-translational) Example: phosphorylation and other protein modifications which occur after the protein has been synthesized can change their activity

51 Noncoding RNAs play multiple roles in controlling gene expression Only a small fraction of DNA codes for proteins, rrna, and trna A significant amount of the genome may be transcribed into noncoding RNAs Noncoding RNAs regulate gene expression at two points: mrna translation and chromatin configuration Effects on mrnas by MicroRNAs and Small Interfering RNAs MicroRNAs (mirnas) are small single-stranded RNA molecules that can bind to mrna These can degrade mrna or block its translation

52 (a) (b) Primary mirna transcript. This RNA molecule is transcribed from a gene in a nematode worm. Each double stranded region that ends in a loop is called a hairpin and generates one mirna (shown in orange) Regulation of gene expression by mirnas. RNA transcripts are processed into mirnas which prevent expression of mrnas containing complementary sequences. Hairpin mirna mirna Hydrogen bond Dicer 1) An enzyme cuts each hairpin from the primary mirna transcript 2) A second enzyme called Dicer, trims the loop and the single stranded ends from the hairpin, cutting at the arrows. 3) One strand of the double stranded RNA is degraded; the other strand (mirna) then forms mirnaprotein a complex with one complex or more proteins 4) The mirna in the complex can bind to any target mrna that contains at least 6 bases of complementary sequence. 5) If mirna and mrna are complementary all along their length, the mrna is mrna degraded Translation blocked degraded (left); if the match is less (b) Generation and function of mirnas complete translation is blocked (right) The phenomenon of inhibition of gene expression by RNA molecules is called RNA interference (RNAi). This is caused by single-stranded small interfering RNAs (sirnas) and can lead to degradation of an mrna or block its translation sirnas and mirnas are similar but form from different RNA precursors

53 Chromatin Remodeling and Silencing of Transcription by Small RNAs sirnas play a role in heterochromatin formation and can block large regions of the chromosome Small RNAs may also block transcription of specific genes