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BIO 220 8. Gene Expression Inheritance At one time scientists thought proteins were the genetic information found in organisms. Later it was found to be DNA. Nucleic Acids and Nucleotides Nucleotides are composed of a nitrogen base, ribose sugar and a phosphate group. A polymer of nucleotides forms a Nucleic Acid or polynucleotide. 2 forms of nucleic acids found in the cell are DNA and RNA. DNA Deoxyribonucleic acid 4 nucleotidesAdenine (Purine) Guanine (Purine) Cytosine (Pyrimidine) Thymine (Pyrimidine) Base Pairing between strands of DNA A-T (2 hydrogen bonds) G-C (3 hydrogen bonds) DNA DS polymer of nucleotides in anti-parallel arrangement. Helical shaped ( helix), right handed helix in the cell. Helix creates Major Groove and Minor Groove in DNA molecule Many DNA binding proteins read exposed bases in major groove. Some proteins may bind minor groove sequences. Inside the cell DNA is supercoiled (twisted about its axis). Supercoiling is achieved by DNA Gyrases or Topoisomerases. Topoisomerase II functions by making a DS break and resealing in DNA to result in a twisting of the molecule. Positive Supercoiling occurs when the DNA strand is twisted in the same direction as the right handed helix. Negative Supercoiling occurs when the twist is in the opposite direction of the right handed helix.


DNA may have addition structure depending on the sequence Bent DNA- string of 5 or 6 adenine residues separated by 4 or 5 bases Bending of DNA involved in regulatory processes. Stemloop Structure- created by inverted repeats. Cruciform structure forms from hydrogen bonding between a single DNA strand. Structure serves a protein-binding site. DNA is chemically the same molecule in Prokaryotes and Eukaryotes but exists in different forms between these groups of organisms. Eukaryotes Linear Wrapped around histones (forms nucleosomes) Found in the nucleus Prokaryotes Circular molecule Packaging lacks histones Found in cytoplasm DNA is viewed using a 5 -> 3 orientation. Composed of coding and noncoding regions Coding Regions (Genes) DNA sequence which encodes the genetic information for a particular protein or structural RNA. Noncoding Regions (many examples) Promoters- upstream DNA sequences which activate expression of a gene. Promoters range from strong to weak. Strong promoters are positioned in front of genes that are expressed frequently (example- genes encoding enzymes involved in metabolism). Weak promoters may activate genes that are expressed in special situations (example- sporulation genes). Enhancer Elements- cis acting sequences upstream or downstream from a gene. Transcription Factors bind to such sequences to affect the strength of a particular promoter. Origin of Replication- Sequence recognized by replication enzymes. Telomeres- terminal ends of linear DNA molecules.


How is the genetic information in DNA expressed? Central Dogma DNA -> mRNA -> Proteins

DNA is the blueprint, i.e. molecular instructions while mRNA contains a temporary version of the information encoded in the DNA. Proteins are the manifestation of the information contained within the gene. Gene arrangements -One gene for one protein hypothesis (eukaryotes)- Now modified -Regulons (prokaryotes) -Overlapping genes (viral systems) ribosome wobble weak ribosome landing pad Transcription The process of making an RNA version of the information contained in DNA. RNA is less stable than DNA. As a result RNA transcripts are only temporary in nature and degrade rapidly in the cell. (Q: What is the utility of making a temporary version of genetic information?) Transcription has many steps which can be divided into 3 broad categories. Initiation Elongation Termination

Steps of transcription


An enzyme RNA Polymerase (RNA POL) slides along (or reads) DNA until it finds a region for which it has high affinity. Enzyme stops and binds by forming hydrogen bonds. This region is called a Promoter. Promoters are sequences upstream of the gene to be transcribed. Promoters contain a Consensus Sequence, a sequence of nitrogen bases that are common among different promoters (example Pribnow box and 35 sequence in Eukaryotes). Identification of promoters is often aided by proteins called Transcription factors. Phosphorylation of RNA Polymerase II in Eukaryotes also affects the activity of the enzyme.

In Bacteria RNA POL helps recognize a promoter with assistance from many accessory proteins including a well-studied protein called Sigma Factor. 70 is the most common form of Sigma Factor in E. coli however 7 different Sigma Factors have so far been identified. Each Sigma Factor helps RNA Pol identify (and thus transcribe) a different subsets of genes. In Eukarya the major subclasses of RNA are transcribed by different RNA Polymerases. RNA Polymerase I- most types of rRNA RNA Polymerase II- all mRNA RNA Polymerase III- tRNA (and 1 rRNA)

2. RNA Polymerase unwinds a portion of the DNA double helix. Complimentary ribose nucleotide triphosphate molecules hybridize to exposed strand.

Initiation- RNA POL begins to travel exposed strand and synthesize the complimentary RNA molecule in a 5 to 3 manner. Initiation occurs with the synthesis of a dinucleotide. Elongation- RNA POL polymerizes nucleotide monomers into a RNA polymer. Proteins called Elongation Factors assist in this step.


5. As the enzyme moves along the DNA, RNA POL re-zips DNA.

Termination- RNA POL encounters a second type of consensus sequence telling it to stop polymerizing RNA. Termination of transcription is caused by the DNA sequence.

Different types of termination signals Inverted repeat followed by AAA



RNA folds into a stemloop structure which derails RNA Pol GC rich regions followed by AT rich regions Rho dependent termination Rho (protein) binds RNA Moves along molecule until it reaches RNA POL RNA Pol pauses at Rho dependent termination site Rho causes RNA POL to fall off DNA thus ending transcription. Transcription produces 3 main types of RNA mRNA tRNA rRNA

___________________________________________________________ mRNA- Messenger RNA Contains an RNA version of the DNA found within a gene The information in mRNA is directly translated into a protein. mRNA often goes through a maturation process before it is functional. Precursor mRNA are often called Primary Transcript. Differences exist between prokaryotic and eukaryotic mRNA including 5 capping, Poly A tail, splicing and the number of genes transcribed on a given mRNA molecule. 5 capThe 5 end of Eukaryotic RNA contains a modified Guanosine residue which delays degradation of the mRNA transcript (or adds stability). Poly A tail The 3 end of Eukaryotic mRNA contains 100-200 residues of the nucleotide Adenine. Enhances export of mRNA from the nucleus Prevents degradation in cytoplasm May enhance translation Splicing5


Not all segments of Eukaryotic mRNA (and some Bacteria RNA) contain a translatable message. Introns- untranslated regions Exons- translated regions In the nucleus (spliceosome) splicing occurs whereby introns are removed and exons are linked to form a mature mRNA molecule. Spliceosome is comprised of proteins and snRNA. Catalytic activity of spliceosome is due to snRNA, not protein component. Catalytic RNA is also referred to as Ribozymes. See Figure 7.30, 7.32 Eukaryotic splicing creates a lariat structure out of intron. Tetrahymena splicing creates a circular intron and a small 15 nt intron fragment. Tetrahymena ribozyme requires a Guanosine residue and Mg++ for catalysis. All ribozymes require metal ions either for catalysis or tertiary folding. Enzymes (or ribozymes) which require metals for catalysis are called Metaloenzymes. Other well characterized ribozymes include the Hairpin Ribozyme, Hepatitis Delta Virus Ribozyme and the Hammerhead Ribozyme. As ribozymes are often contained in introns they can be referred to as Self-Splicing Introns.

Polycistronic RNA Eukaryotic mRNA generally contains 1 gene per mRNA molecule (monocistronic RNA). Prokaryotes tend to have related genes i.e. genes involved in the same biochemical pathway close together on their DNA. As all such genes are required at the same time, the genes are transcribed on a single mRNA, referred to as Polycistronic RNA.

tRNA- Transfer RNA



Unlike mRNA, tRNA lacks a genetic message but instead acts as a structural element involved in the translation of mRNA sequences. Short molecule (73-93 nucleotides long) Contains 4 stems and 3 loops Interacts both with Amino Acids and mRNA Draw molecule on board Because tRNA have specific anticodon sequences, a specific tRNA exists for every codon. 100-110 species of tRNA in Eukaryotes 60 species of tRNA in Bacteria Codon- sequence of 3 nucleotides in an mRNA molecule which calls for a specific amino acid to be incorporated into a growing protein. Genetic Code- Composed of Nucleotide triplets specifying for a particular amino acid (codon). Redundant more than one triplet may code for the same amino acid. Example- ACU, ACC, ACA all encode for Threonine. All codons for a particular amino acid may not be used equally; one codon may be the preferred sequence. For example, in organism X ACU may be the preferred codon for threonine but in organism Y ACC may be preferred. Such a situation is called a Codon Bias and must be taken into account when moving genes from one organism to another. See Table 7.3 Unconventional Amino Acids Selenocysteine- the 21st amino acid UGA is normally a stop codon but sometimes encodes for selenocysteine. Choice depends on secondary structure of mRNA and flanking sequences. Selenocysteine is found in both prokaryotic and eukaryotic organisms. Pyrrolysine UAG is normally a stop codon but sometimes encodes for pyrrolysine. Choice depends on secondary structure of mRNA and flanking sequences. First isolated in certain methanogens.


Charging of tRNA Covalent linkage of tRNA and Amino Acid (AA) Involves 2 chemical reactions in cytoplasm Catalyzed by the enzyme Amino Acyl tRNA Synthetase Specific Amino acyl tRNA synthase for each codon AA/combination Enzyme recognizes the anticodon region of the tRNA and the amino acid AA + ATP -> AMP-AA + P-P tRNA + AMP-AA -> tRNA-AA + AMP + 2 Pi

How are charged tRNA molecules processed according to the information in mRNA to make protein? Involves interaction between mRNA, tRNA and Ribosome Known as Translation

RibosomeComplex of proteins and rRNA (Ribosomal RNA) Consists of 2 subunits- Small subunit and Large subunit Ribosome Characteristics Property Overall Size Small Subunit # Proteins Small su Prokaryote 70s 30s ~21

Eukaryote 80s 40s ~30


RNA Size Large Subunit # Proteins Large su RNA Size

16s 50s ~34 23s, 5s

18s 60s ~50 28s, 5.8s, 5s

Differences in Prokaryotic and Eukaryotic ribosomes have implications in antibiotic therapy against protein synthesis. rRNA Contains no genetic message Acts as a structural and catalytic element in the translation of mRNA into protein. 16s rRNA- helps 30s sub unit identify Shine Delgarno sequence of mRNA. 16s rRNA- physically attaches tRNA via anti-codon loop to ribosome. 16s rRNA- participates in recognition of Release Factor proteins. 23s rRNA recognized acceptor stem of charged tRNA 23s rRNA- catalyzes formation of peptide bonds 23s rRNA- aids in translocation Translation- translating the message in mRNA into protein Initiation Elongation Translocation Termination

Initiation (Prokaryotes) 30 s (small subunit) binds to docking site (Shine Delgarno Sequence) present in mRNA with help of Initiation Factors. 16s rRNA hybridizes to SD sequence. tRNAMET binds to 30s s.u. and mRNA This step requires energy in the form of high energy phosphate bonds contained within GTP.


tRNAmet is the first AA in eukaryotic protein synthesis (formyl-methione in prokaryotes) Formyl group or entire amino acid may later be removed in posttranslational modifications. Initiation Factors (proteins) recruit 50s (large) sub unit. At this point the intact ribosome has been formed with tRNAmet in P site. Intact ribosome contains 3 sitesA site (acceptor site) P site (peptide site) E site (exit site) Show drawing on board tRNAmet (initiation codon) found in what is referred to as the P site (peptide site). GUG may sometimes serve as alternative initation codon. Formylmethionine initiator tRNA is found in the P site upon completion of the intact ribosome (not valine). Elongation With tRNAmet in the P site, the tRNAAA signified by the next codon (RNA triplet) hybridizes to the mRNA. (For example, lets say the next codon in the mRNA is GGG encoding for Glycine.) This bonding occurs via hydrogen bonding between the codon in mRNA and the anticodon sequence found in the tRNAgly. RNA catalyzed condensation reaction creates dipeptide attached to tRNAgly-met. Elongation requires GTP and elongation factors (proteins) Proteins are synthesized from the amino terminus (or N terminus) to the carboxy terminus (or C terminus). Thus covalent linkage occurs between the C terminus of the nascent peptide and the amino group of the next amino acid added to the chain. At this point in the process the dipeptide linked to tRNAgly-met is contained in the A site while the tRNA which was linked to methionine is now uncharged but still located in the P site.


Translocation Ribosome moves along mRNA GTP and elongation factors are required The dipeptide tRNAgly-met now occupies the P site of the ribosome. A site is now vacant, ready for the next tRNA molecule specified by the codon in the mRNA. E site contains uncharged tRNA (once linked to methionine). During subsequent translocation uncharged tRNA once linked to methionine discharged from the ribosome. Termination Release Factors enters A site and binds to nonsense or stop codon Release Factors hydrolyzes tRNA-protein bond, releasing protein Ribosome dissociates into subunits + mRNA

Polysomes Multiple ribosomes translating same mRNA molecule SRP- a short peptide leader called a signal sequence may signify the secretion of a protein or the insertion of that protein into the membrane. A Signal Recognition Particle (SRP) compose of protein and RNA binds the signal sequence and facilitates entry of the growing peptide into the membrane bound transport protein. This is the system utilized with Sec translocases (secYEG for example.) These proteins require ATP to translocate peptide. Often the SRP recognized leader sequence is removed upon completion of protein export. An alternative system called Tat (twin arginine translocase) protein export system moves a protein across the membrane after it has been properly folded. Tat protein export uses the Proton Motive force to provide the energy required for export.



Proper FoldingAll proteins must be folded in the proper confirmation to be active. Chaperonins may assist in this process. DNA J and DNA K are ATP dependent chaperonins. They function in the cell to prevent a protein from folding too quickly 9and possibly improperly.) GroEL and GroES are also ATP dependent chaperonins. These proteins may help fold proteins that DNA J and DNA K were unable to fold. They also may help re-fold proteins that are partially denatured. Post translational ModificationsNot all proteins are functional upon the completion of translation. For example- signal sequences may be removed, proteins may be phosphorylated, disulfide bridges may form, adenylation at certain sites or glycosylation may be required to form a functional protein. Any such change which occurs upon the completion of translation is called a Post Translational Modification. Such covalent modifications may function to serve as a regulatory step. For example Glutamine Synthase can be progressively adenylated (up to 9 times). Each adenylation decreases the overall activity of the enzyme. (The enzyme can be activated by removal of the adenyl groups). See Figure 8.6 Inteins and Exteins Protein splicing has been observed, analogous to mRNA splicing in Eukaryotes where certain regions are removed (Inreins) and other regions are covalently linked together (Exteins) to form the mature protein. DNA Gyrase subunit A in Mycopbacterium leprae undergoes such processing. (See Figure 8.7)

Removal of Leader Sequences Often the first amino acid (formyl methionine) is removed as part of post translational modifications. Sometimes a N terminus leader sequence (such as that required for secretion) is removed.

Control of Gene Expression Occurs at several levels



Initiation of transcription Elongation of transcription Initiation of translation Post translational modification

DNA Binding Proteins- Motifs Proteins that interact with DNA often have characteristic motifs that allow the protein to recognize specific sequences in the DNA. Most DNA binding proteins read the Major Groove while some read the Minor Groove of the molecule. Helix Turn Helix motif contains a stabilizing helix and a recognition helix. Often HTH proteins exist as dimmers with interaction occurring between two stabilizing helices. Leucine Zipper proteins are an example of HTH proteins. The two stabilizing helices interact via hydrophobic interactions between leucine residues spaced every 7 amino acids. Zinc Finger motif proteins contain a DNA recognition helix that is stabilized by 2 histadine residues in the helix and 2 cysteine residues in another part of the protein. The 4 amino acids coordinate to a Zn++ ion. The resulting finger is this kept stable to read DNA. Regulation of Transcription Initiation Negative Control involves operons and Repressor proteins Lac Operon- inducible Diptheria toxin gene- inducible Trp Operon- repressible Positive Control- involves Activator proteins Catabolite Activator Protein (CAP) + cAMP Operons and Regulons Operon-Series of genes whose end products function together in a pathway Linked by a single promoter Contain on/off switch called an Operator Operator is located between promoter and genes


Regulon- a series of operons controlled by a single regulatory protein Show diagram on board Lac OperonContains genes to metabolize lactose Lactose- disaccharide of glucose + galactose Normally off- Inducible Repressor binds operator, prevents transcription (RNA pol cant proceed) In the presence of inducer lactose, operon is on. Lactose binds with repressor, allosteric changes make repressor inactive. RNA pol proceeds (Allosteric regulation refers to a molecule binding a protein in a site other than the proteins active site. Binding results in confirmational change to active site making it either favorable to bind substrate (allosteric activation) or less likely to bind substrate (allosteric inhibition).) Identification of Lac mutants shed light on this process

TRP Operon Normally TRP operon is always on, transcribing the enzymes involved in typtophan biosynthesis. However in the presence of excess tryptophan, operon is shut off. Process is said to be repressible. Tryptophan binds to repressor causing allostreric changes which allow repressor to bind operon. Transcription is stopped. Tryptophan acts as corepressor.

Diptheria Toxin Expression of the gene encoding for the diphtheria toxin is regulated by Fe3+. Iron binds to repressor protein resulting in an allosteric


change whereby the repressor is now able to bind DNA and prevent transcription. In the absence of Fe3+ the repressor is unable to bind the operator and transcription proceeds. Why would a toxin be regulated by the presence or absence of iron?

Catabolite Repression Not all energy sources are equally preferred by the cell, thus certain catabolic pathways are not always turned on. This is referred to as Catabolite Repression. The Glucose Effect Catabolite Repression was first observed with cultures grown in mixed nutrient broths containing glucose and lactose. Glucose, being the preferred energy source was utilized first. Upon the depletion of glucose, growth curves enter a lag phase. Later growth resumes as lactose catabolic pathways are expressed. This pattern of growth is called Diauxic Growth. Catbolite repression is inactivated by the activator protein, Catabolite Activator Protein CAP, (sometimes called cAMP Binding Protein or CRP). In the cell, glucose is the preferred energy source. As glucose levels drop, so do corresponding levels of ATP. As a result, a secondary metabolite cAMP (cyclic adenosine monophosphate) begins to accumulate. cAMP binds to CAP, resulting in allosteric changes which allow CAP to bind DNA near a promoter region. CAP attracts RNA POL to the promoter allowing transcription of the gene to occur. In the absence of CAP-cAMP complex, RNA POL will not bind to the promoter.

Alternate Sigma factors (Regulation at the level of Transcription) 7 different Sigma Factors in E. coli. Amount of each factor controls transcription of various genes. 70 controls transcription of most genes.


Other sigma factors control genes involved in Nitrogen Assimilation, Stationary Phase, Osmotic Response, Flagella Synthesis.

Regulation at the level of Elongation of Transcription Examples- Tryptophan biosynthesis Tryptophan Biosynthesis The TRP Operon contains a short Leader Sequence containing tryptophan residues. Translation of the 5 end of an mRNA from the TRP operon occurs before transcription is complete, or during the Elongation phase of transcription. If the cell is tryptophan starved, translation of the leader sequence cannot occur quickly. Untranslated mRNA located between the ribosome and RNA POL folds into a stem-loop structure which allows transcription to proceed. If the cell contains excess tryptophan, translation of the leader sequence occurs relatively quickly and the untranslated mRNA between the ribosome and RNA POL folds into an alternative stem-loop structure which derails RNA POL and terminates transcription.

Regulation at the Initiation of Translation Riboswitches Interactions between 5 untranslated region and Poly A tail Riboswitches mRNA may fold into alternate structures, especially at the 5 untranslated region of the molecule. Such confirmations often involving the binding of a metabolite (which the biosynthesis for is encoded by the mRNA) to the mRNA. In such instances binding results in a confirmation which prevents translation. This is an example of Negative Feedback seen at the RNA level. In the absence of the metabolite the mRNA folds into a confirmation which allows translation to proceed.



Natural Riboswitch Targets: Coenzyme B12 Thiamine pyrophosphate FMN S-adenosylmethionine Guanine Adenine Lysine

5 UTR and Poly A tail interactions The Poly A tail of mRNA can interact with PABP (Poly A Binding Protein) which in turn interacts with Initiation Factors involved in ribosome assembly. The entire structure takes on the structure of a closed loop which stabilizes the mRNA and increases translational efficiency.

Small RNA regulation of gene expression- SiRNA and MicroRNA SiRNA -Antisense RNA Involves regulatory RNA (as opposed to Regulatory Proteins). A source of Antisense RNA is a small gene with identical sequence to the gene to be regulated, but having its promoter at the 3 end of the gene. When this gene is transcribed, an RNA complimentary to the mRNA of the regulated gene is expressed. This small RNA, called Antisense RNA will hybridize to mRNA and form a double stranded RNA complex which is not translatable. SiRNA (small interfering RNA) forms the basis of a new type of therapy RNAi (RNA interference.) SiRNA is used to study the roles of various gene products and is also being used in clinical trials as a form of medicine, notably SiRNA against macular degeneration.

MicroRNAThese small RNA molecules have recently been shown to affect gene regulation, however the mechanisms are not as straightforward as those with SiRNA. MicroRNA fold into a stem loop structure for stability then interact with various proteins to form a regulatory structure. Some MicroRNA will degrade mRNA while others will merely prevent translation of the mRNA. A single MicroRNA has the ability to


regulate expression of whole classes of genes and complex processes such as the Cell Cycle. let7 gene encodes for a microRNA molecule that plays a role in cell differentiation of C. elegans and allows cells to exit the Cell Cycle. Cells lacking let7 microRNA cannot exit the cell cycle and proliferate like tumor cells. Human lung cancer cells lack let7 microRNA.

Signal Transduction (Post translation Modifications) Signal Transduction is the process by which an external signal results in intracellular changes/responses. The external signal does not enter the cell. Often these processes involve 2 proteins and are called the Two Component System. This system is used in cellular response to chemotaxis. Two Component System involves Sensor Protein Response Regulator Protein Sensor Protein Membrane bound Binds signal molecule Binding results in autophosphorylation at Histidine residue. Also called a Sensor Kinase.

Response Regulator Protein Becomes phosphorylated by Sensor Kinase Activated protein may bind DNA as activator, repressor or serve other function. Two Component System becomes terminated by Phosphatase. Phosphatase removes phosphate from Response Regulator Protein. Metabolic Regulation



Allostery involves changes in shape of an enzyme that affects its activity. Allosteric Effector molecules can bind to an Allosteric site of the enzyme resulting in a conformational change that either activates or inhibits the enzyme. Feedback Inhibition results when the end product of a metabolic pathway acts as an Allosteric inhibitor of an enzyme that catalyzes a step earlier in the pathway. Isoenzymes are different enzymes that catalyze the same reaction. (See Figure 8.5). Aromatic amino acid synthesis involves a precursor step catalyzed by the enzyme DAHP Synthase. 3 different isoenzymes of DAHP Synthase are allosterically inhibited by different aromatic amino acid end-products. The 3 amino acids (Tyrosine, Phenylalanine and Tryptophan) participate in Concerted Feedback Inhibition to regulate the total cellular activity of DAHP Synthase.

DNA Replication Question: By what manner does DNA replication occur? 3 theoriesConservative Semi-conservative Dispersive Meselson/Stahl data indicates semi-conservative replication Origin of Replication 300 bp section of DNA where initiation of replication occurs Sequence specific Multiple origins of replication Replication occurs bidirectionally Process of DNA Replication 1. Origin sequence recognized by Origin Binding Protein. 2. DNA unwinding by Helicase (ATP dependent). 3. Unpaired DNA stabilized by Single Strand Binding Proteins. 4. Primase generates RNA primer 5. DNA Polymerase III (DNA Pol III) synthesizes new DNA strand 5 to 3 6. RNA primers removed by 5 to 3 exonuclease activity of DNA POL I 7. RNA Primers replaced by 5 to 3 polymerase activity of DNA POL I 8. Nicks are sealed by DNA Ligase


8. DNA Gyrase (Topoisomerase) supercoils DNA into supercoiled structure Features; Replication forks Okzaki fragments Theta Structures Leading Strand- continuous synthesis, 5->3 Lagging Strand- discontinuous synthesis 5->3 Replisome Replisome- A replication factory Protein:DNA complex which allows fluid replication of leading and lagging strands. Looping out of lagging strand occurs through DNA interaction with proteins. Replisome contains Helicases, 2 DNA POL III and Primase! Rolling Circle replication Seen in replication of some plasmids, SS DNA viruses, replication of satellite RNA. DS DNA model1. Nick initiates formation of SS DNA template 2. Circular molecule appears to roll away from linear template 3. Linear DNA and circular strand replicate separately