Modification of Genes and Proteins

Modification of Genes and ProteinsBy Paul Southard, Joshua Pikovsky, and Jake SecorTranscript ProcessingProtein FoldingRNAiGene Repair

Genes and proteins are modified by transcript processing, protein folding, RNA interference, and gene repair.2Transcript ProcessingIntroduction to Transcript ProcessingTranscription factor recognizes TATA Box and binds to DNARNA polymerase bonds to DNARNA polymerase separates strands and strings together complementary nucleotides (using U instead of T)Primary transcript has been created when terminator region is reached

In transcript processinga transcription factorrecognizes aTATA Box(nucleotide sequence with sequential TATA) and binds to the DNA. The TATA Box is generally a few dozen nucleotides upstream from the starting point of the transcription region. The TATA box and subsequent nucleotides until the start point is known as thePromoter Region.Once transcription factors are bound,RNA Polymerasecan bond to the DNA. The combination of the two is called theTranscription Initiation Complex. The RNA Polymerase separates the strands and strings together nucleotides that are complementary to the DNA template strand. The only difference between these and the non-template strand is that, in RNA, U nucleotides are used instead of T nucleotides. The RNA Polymerase moves down the template strand, unwinding the double helix and continuing to string together nucleotides until it reaches the terminator region. By the end of this process, the cell has created the primary transcript.4Introduction to Transcript Processing

Transcription:Creates molecule to carry protein instructions from DNACreates exact replica complementary to DNA

Transcription is a process intended to create a molecule that can carry the exact message of DNA to the parts of the cell where proteins are made. It occurs in the nucleus and its end product is theprimary transcript. It creates an exact replica by using many proteins to string together a single strand of nucleotides complementing those of the DNA. Here is an animation describing how mRNA is processed.5Transcript ProcessingAlteration of ends of transcript:5 end capped with modified guanineKeeps RNA from degrading in the cytoplasmCleavage factors and stabilizing factors bind to 3 endPoly A polymerase binds and cleaves 3 end and adds poly A tail made of adenine

When the transcript is altered, the 5' end gets capped with amodified guanine nucleotide. This happens as soon as transcription starts. This cap keeps the RNA from degrading in the cytoplasm and helps the ribosome know where to start. Once transcription ends, twoCleavage Factorsbind to the 3' end along with twostabilizing factors, at which pointpoly A polymerasebinds and cleaves the end. Poly A Polymerase then puts apoly (A) tailon the 3' end. This consists of 50-250 adenine nucleotides. This serves the same function as the 5' end. The poly (A) tail may also make it move more easily.6Transcript ProcessingRNA splicing:Nucleotides removedIntrons = non-coding regionsExons = coding regions to be expressedSmall nuclear ribonucleoproteins (snRNPs) = proteins that detect adenine at branching siteSpliceosomes remove the intron and bind the two exons

A lot of nucleotides are removed from the transcript in the process ofRNA splicing.The majority of the RNA is noncoding, and must be removed. Noncoding regions are calledintrons, coding regions that will be expressed are called exons. Ends of introns are marked by nucleotide sequences. There is a GU at the5' splice siteand an AG at the3' splice site.There is also aBranch sitein the middle that consists of one adenine. These can be detected by proteins calledsmall nuclear ribonucleoproteins(snRNPs). snRNP contains asmall nuclear RNA(snRNA). The snRNPs are parts of largerspliceosomesthat act on the splice sites. They cuts these sites, remove the intron and bind the two exons together where the splice sites were. In one specific example, a U1 snRNP binds to the 5' site, a U2 binds to the Branch site and the U3, U4 and U5 bind to the rest of the intron, comprising one spliceosome. First, the 5' end is cut, which curls up and connects to the adenine of the branch site. After that the 3' end is cut, and the snRNP's dissociate.7

Protein FoldingIntroduction to Protein FoldingThe sequence of amino acids defines a proteins primary structure.

A protein is made up of a collection of amino acids. A proteins structure is defined by its amino acid sequence. Because the amino acid sequence also determines a proteins function, structure is crucial to function and vice versa.10Introduction to Protein FoldingBlueprint for each amino acid is characterized by base tripletsFound in the coding region of genesRibosomes recognize triplets and create proteins

Base triplets are sets of three letters that characterize each amino acid. They are found in the coding regions of genes. Triplets are recognized by ribosomes which create proteins. In its primary structure, a protein is in the form of a linear amino acid sequence. After its primary structure, a protein takes on its secondary structure, usually a pleated sheet and alpha helix. The protein only becomes functional when it is folded into its three-dimensional, tertiary structure. A proteins tertiary structure cannot be determined just from its gene sequence.11

These diagrams demonstrate how an amino acid chain folds to form its tertiary structure. It is not known how an amino acid chain folds into its tertiary structure in fractions of a second. Folding, along with intramolecular bonds created by the initial amino acid sequence, determines the proteins tertiary 3D structure. When a protein folds it tests multiple conformations and shapes before reaching its unique and compact final form.12Protein FoldingCovalent bonds between amino acids help stabilize the proteinShape and stability also maintained by chemical forces

Proteins that are folded are kept stable by thousands of noncovalent bonds between the amino acids. Chemical forces between a protein and its environment also contribute to the shape and stability. For example, proteins that are dissolved in the cytoplasm have hydrophilic chemical groups on their surfaces, while hydrophobic elements are tucked inside.13Protein FoldingChaperone proteins:Prevent nearby proteins from inappropriately associating and interfering with proper foldingSurround protein in protective chamber during foldingEx) bacteria: GroEL and GroESUse ATPAlso assist in refolding proteins

Due to the crowded nature of cytoplasm, cells rely on chaperone proteins to prevent nearby proteins from inappropriately associating and interfering with proper folding. Chaperone proteins surround a protein during the folding process. For example, in bacteria, many chaperone GroEL form a hollow chamber over proteins while they are folding. Molecules of a second chaperone, GroES, form a lid over the hollow chamber. Chaperones are common in cells and use ATP to bind and release polypeptides as they fold. Chaperones also help refolding proteins, for folded proteins are surprisingly fragile and can easily denature, or unfold, due to subtle increases in temperature or changes in environmental conditions. Refolding proteins is important because it is much more efficient to fix an unfolded protein than to synthesize an entire new protein.14Protein Folding

Chaperone proteins protecting folding proteins

Some protein folding occurs during translation, but most occurs in the endoplasmic reticulum. Protein molecules fold spontaneously during or after synthesis, and while it is a mostly independent process, it relies on the solvent (water or lipid bilayer), salt concentration, temperature and availability of chaperone proteins.15Protein FoldingModels of protein folding:Diffusion Collision Model:Nucleus is formedSecondary structures collide and pack togetherNuclear Condensation Model:Secondary and tertiary structures are made simultaneously

There are two models of protein folding. The Diffusion Collision Model states that a nucleus is formed, then secondary structures collide and pack together. The Nuclear Condensation Model involves secondary and tertiary structures that are made simultaneously.16RNAiIntroduction to RNAiRNAi = RNA InterferenceRNAi is used to:Silence specific genesFix gene expression problems in mammals

Also known as:CosuppressionPost Transcriptional Gene SilencingQuelling

RNA interference, or RNAi, is the process used by cells to turn off certain genes. RNAi is the fastest known way to silence specific genes, and also fixes gene expression problems in mammals.18Introduction to RNAiTypes of small silencing RNA:Small interfering RNA (siRNA)Endogeneous: derived from cellExogeneous: delivered by humansMicro RNAs (miRNA)PIWI-interacting RNAs (piRNA)RNAi breaks up mRNA before it is synthesized.

There are three types of small silencing RNA. Small interfering RNA, or siRNA, is derived from double stranded RNAs either endogeneously or exogeneously, as shown in the picture below, which will reappear shortly. SiRNA regulates gene expression. Micro RNA, or miRNA, comes from RNA transcribed in the nucleus. In order to effectively stop proteins from being synthesized, RNAi molecules must break up mRNA before it is synthesized.19Introduction to RNAi

The pictures below show how the cell cleaves and degrades the mRNA selected by the RNAi.20Introduction to RNAi

This video gives a simplified explanation of how RNAi functions.21Implications of RNAiAllows singling out of genes to determine function.

When an individual gene is turned off through RNA interference, phenotypic observations can be made to determine what function that specific gene has. For example, if a purple flower turns white when gene one is turned off, then gene one relates to color. If petals stop growing when gene two is turned off, then gene two controls petal production.22Implications of RNAiCould halt progression of:CancerHIVArthritisAll other diseases

Because RNAi stops production of proteins, harmful diseases like Cancer, which currently have painful, life-consuming effects and treatments, can be defeated.23Implications of RNAi

If RNA, modified to be identified as a disease and coded for the production of cancerous proteins, is added to a cell that is already producing those proteins, then the RNAi in the cell will kill not only the added cancer RNA, but all other RNA signaling the production of cancer proteins. Production of these proteins would be completely stopped; effectively halting cancer in the cell.24Jean RepairIntroduction to Jean Repair

Gene RepairIntroduction to Gene RepairDNA can be damaged by:Radiation (gamma, x-ray, and ultraviolet)Oxygen radicals from cellular respirationEnvironmental chemicals (hydrocarbons)Chemicals used in chemotherapy

There are a variety of external and internal factors that can damage DNA. Radiation is harmful, especially among gamma, x-ray and ultraviolent wavelengths. Some oxygen byproducts from cellular respiration are dangerous as they are highly reactive. Various environmental chemicals, particularly hydrocarbons (found in cigarette smoke) can be harmful as they cause serious mutations in DNA. Chemicals used in chemotherapy are also capable of damaging DNA.28Introduction to Gene RepairFour major types of DNA damage:Deamination: amino acid group lostMismatched baseBackbone breakCovalent cross-linkage between bases

Deamination in DNA

There are four major types of possible DNA damage. The first is deamination, which is essentially when an amino group is lost. This can be responsible for converting a C base to a U. The second is the mismatch of a base as a result of a proofreading failure during DNA replication. One of the more common examples of this is the incorporation of U instead of T. Next is the backbone break, which can be limited to one of the two strands of DNA or both strands. The most common cause of this is ionizing radiation. The fourth and last major type of DNA damage is the covalent cross-linkage between bases. This can occur on the same DNA strand (intrastrand) or on opposite strands (interstrand).29Gene RepairRepairing damaged bases:Direct chemical reversalExcision repair mechanisms:Base excision repair (BER)Nucleotide excision repair (NER)Mismatch repair (MMR)

There are four primary mechanisms for repairing damage to DNA. The first is direct chemical reversal, often through enzymes. Direct chemical reversal deals with specific problems. More general repairs are done by excision repair mechanisms. These repairs are classified under base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR).30Gene RepairChemical ReversalEx) glycosylase enzymes remove mismatched T and restore correct C

One of the most frequent causes of point mutations is a spontaneous bonding of a methyl group to a cytocine base after it is removed from a T. These are easy to repair, as glycosylase enzymes remove the mismatched T and restore the correct C. While this does solve the problem, it isn't efficient because each of the various problems requires a specific mechanism to be fixed.31Gene RepairExcision repair mechanisms:Base excision repair:DNA glycosylases identify damaged basesDNA glycosylases remove damaged basesDeoxyribose phosphate backbone component removed, creating gapGap filled with correct nucleotideBreak in strand ligated

The first of the excision repair mechanisms is base excision repair. Base excision repair has a few steps. First, DNA glycosylases identify and remove damaged bases. Next, the deoxyribose phosphate backbone component is removed, creating a gap. Then, it is replaced with the correct nucleotide, relying on DNA polymerase beta, one of more than 11 DNA polymerases encoded by our genes. Finally, the break in the strand is ligated, or bound, requiring two ATP reliant enzymes.32Gene RepairExcision repair mechanisms:Nucleotide excision repair:Protein factors identify damageDNA is unwoundFaulty area is cut out and the bases are removedDNA is synthesized to match that of the opposite, correct strandDNA ligase adds synthesized DNA

The next method of excision repair is nucleotide excision repair. Nucleotide excision repair uses different enzymes, and instead of removing just one incorrect base, it takes a whole patch of adjacent bases. First the damage is identified by protein factors. The DNA is unwound, creating a bubble like shape using an enzyme system (Transcription factors IIH, TFIIH). Cuts are then made on both sides of the faulty area, and the bases are removed. DNA synthesis using the opposite, correct strand fills in nucleotides. Finally, DNA ligase covalently adds the correct part into the DNA backbone. This can also be coupled with transcription, for it occurs most quickly in cells whose genes are being actively transcribed, or on a DNA strand that is a template for transcription.33Gene RepairExcision repair mechanisms:Mismatch repairCorrects mismatches of normal bases (A&T, C&G) by:Identifying mismatched basesCutting mismatched bases

The third mechanism for excision repair is mismatch repair. Mismatch repair corrects mismatches of normal bases (A&T, C&G). This involves two major steps: theidentificationof a mismatch and the cutting of the mismatch.34

This diagram shows what types of gene damage are repaired by each gene repair method.35Any Questions?