Modification of Genes and ProteinsBy Joshua, Paul, and Jake


external image protein_molecule1.jpg




Transcript Processing

Transcription

  • This process is 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 the primary transcript. It creates an exact replica by using many proteins to string together a single strand of nucleotides complementing those of the DNA.
The Process
  • A Transcription Factor recognizes a TATA Box (nucleotide sequence with sequential TATA) and binds to the DNA. The TATA Box is generally a few dozen nucleotides upstream from the strarting point of the transcription region. The TATA box and subsequent nucleotides until the start point is known as the Promoter Region. One transcription factors are bound, RNA Polymerase can bond to the DNA. The combination of the two is called the Transcription 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 nuclesotides. 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. (1)
Alteration of Ends of Transcript

  • The 5' end gets capped with a modified 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, two Cleavage Factors bind to the 3' end along with two stabilizing factors, at which point poly A polymerase binds and cleaves the end. Poly A Polymerase then puts a poly (A) tail on the 3' end. This consists of 50-250 adenine nucleotires. This serves the same function as the 5' end. The poly (A) tail may also make it move more easily. (2)
Caps.png

RNA Splicing
  • A lot of nucleotides are removed from the transcript in the process of RNA splicing. The majority of the RNA is noncoding, and must be removed. Noncoding regions are called introns, coding regions that will be expressed are called exons. Ends of introns are marked by nucleotide sequences. There is a GU at the 5' splice site and an AG at the 3' splice site. There is also a Branch site in the middle that consists of one adenine. These can be detected by proteins called small nuclear ribonucleoproteins (snRNPs). snRNP contains a small nuclear RNA (snRNA). The snRNPs are parts of larger spliceosomes that 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. (3)
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How the spliceosome cuts the intron out


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RNA Interference

Purpose
  • The process by which RNA strands are injected into a cell, which trigger the cell to break down all messenger RNA for a certain gene. This silences expression of that gene. Other terms for this are cosuppression, Post Transcriptional Gene Silencing (PTGS), and quelling. (8)
  • This mechanism probably evolved about a billion years ago, most likely as a defense against viruses that use RNA. There is a period where the virus's RNA is double-stranded, which would activate RNAi to make siRNAs that would break up mRNA created by the virus so that it didn't turn into harmful proteins. (7)
  • Although miRNA uses many of the same mechanisms as siRNA, it has a different overall purpose, that being to regulate normal, healthy gene expression rather than combat viruses. (7)
  • There is a lot of promise to biomedical researchers in RNAi, because it would allow them to "turn off" certain genes and see what their function is. It could also be used to turn off genes in cells that are making proteins incorrectly and thus harming the cell. (7)
siRNA
  • These regulate gene expression after transcription has occurred, and are one of the main mechanisms of RNA interference. They are derived from double stranded RNA (dsRNA)
    Dicer cleaves an ssRNA into an siRNA duplex
    Dicer cleaves an ssRNA into an siRNA duplex
    made in the nucleus of the cell, which is known as endogenous, or from double stranded RNA delivered by people into cell, which is exogenous. It is important to note that a healthy cell would not create these double-stranded RNA molecules, but that they come generally from RNA viruses. Endogenous dsRNA exits the nucleus through a nuclear pore complex, which pushes it toward ribosomes. In order to prevent mRNA from being synthesized, siRNAs have to break it up in the cytoplasm. (4)
  • A double-stranded-RNA-specific protein called Dicer cuts the dsRNA strand into a segment about 21 nucleotides long. For exogenous dsRNA, an effector protein (RDE-4 in C. Elegans and R2D2 in Drosophila) must detect the dsRNA and activate Dicer activity. The cleaved dsRNA binds to the protein Argonaute and then splits into two ssRNAs (single-strand): the Passenger Strand and the Guide Strand. The passenger strand degrades in the cytoplasm, but the guide strand remains bound to the argonaute protein. The ssRNA and the argonaute together are referred to as the RISC (RNA Induced Silencing Complex). (4)
  • The siRNA base-pairs to target mRNA and the argonaute protein cleaves that mRNA strand. Exonucleases then degrade the mRNA strands. Since the mRNA is degraded, it
    RISC cleaves an mRNA strand, which is then degraded by an exonuclease
    RISC cleaves an mRNA strand, which is then degraded by an exonuclease
    cannot be made into a protein by translation. (4)
miRNA
  • Primary microRNAs (pri-miRNAs) are made in the nucleus and cleaved, at which point they form a precursor microRNA (pre-miRNA) that is 60-70 nucleotides in length. These pre-miRNAs are not double stranded, but are hairpin-like loops with certain parts being double-stranded. These are part of the cell's own genome, and are used to regulate gene expression, not as a defense against viruses. The pre-miRNA binds to dicer the same way that dsRNA does, and is cut into 21 nucleotide segments that bind to argonaute. For miRNAs, only the small seed part base-pairs to the target mRNA. This means that miRNA can target many, many mRNAs, as opposed to the perfectly complementary and specific siRNAs. (4)
The RISC with an miRNA only base-pairs the "Seed", so it can be applied to many kinds of mRNA
The RISC with an miRNA only base-pairs the "Seed", so it can be applied to many kinds of mRNA
Discovery
  • RNAi was first discovered when a startup company was trying to make petunias more purple by injecting an exogenous pigment producing gene. To their great surprise, the introduction of the gene turned the flowers perfectly white. The same thing occured in the worm C. Elegans. (6)
The effect of RNAi on purple petunias
The effect of RNAi on purple petunias











Protein Foldingexternal image protein_folding.jpg


  • A protein's primary, initial structure is defined by sequences of amino acids. The blueprint for each amino acid is characterized by sets of three letters (base triplets). These are found in coding regions of genes and are recognized by ribosomes, which then create the proteins. The resulting protein is a linear chain of amino acids, yet it only becomes a functional protein when it is folded into its three-dimensional structure (Tertiary Structure). Tertiary structures occur after secondary structures, the most common structures of which are pleated sheets and alpha helices. These secondary structures are formed by a small quantity of amino acids in close proximity. These amino acids, once part of the secondary structure, interact, fold, and coil to produce the tertiary three dimensional structure that contain a protein's functional regions (domains).
The three stages of protein folding
The three stages of protein folding

  • A protein’s tertiary structure cannot be determined from gene sequence as of yet, and is also not known how an amino acid chain folds into its tertiary structure in the short time scale (fractions of a second) that occurs in a cell
  • The primary structure of a protein (the initial amino acid sequence) causes the folding and intramolecular bonding of linear amino acid strands, thus determining the unique 3d shape. Hydrogen bonding between amino groups and carboxyl groups in neighboring regions of the protein chain causes certain patters (the pleated beta sheets and alpha helices)
  • When proteins fold, they test multiple conformations and shapes before reaching their unique and compacted final form. These proteins that are in the folding process are kept stable by thousands of noncovalent bonds between the amino acids, along with various chemical forces between a protein and its environment that also contribute to the shape and stability. An example of this is when proteins that are dissolved in the cytoplasm have hydrophilic chemical groups on their surfaces, they keep their hydrophobic parts inside.
  • Due to crowded nature of cytoplasm, cells rely on chaperone proteins to prevent nearby proteins from inappropriately associating and interfering with proper folding. These 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/release polypeptides as they fold. Chaperones also help refolding proteins, for folded proteins are surprisingly fragile/weak and can easily denature (unfold) due to subtle increases in temp, etc, as repairing existing proteins using chaperone proteins is more efficient than synthesis
Chaperone proteins protecting folding proteins
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.
  • There are two models of Protein folding: the Diffusion Collision Model states that a nucleus is formed, then secondary structure and these structures collide and pack together, while the Nuclear Condensation Model involves secondary and tertiary structures that are made simultaneously







Gene Repairexternal image b810008-275-FOR-TRIDION_tcm18-130833.jpg

  • There are a variety of external and internal factors that can damage DNA. Radiation is quite harmful, especially among gamma, x-ray and ultraviolent wavelengths. Oxygen radicals that come as a byproduct of 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 the DNA. Chemicals used in chemotherapy are also capable of damaging 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 (a single strand break, SSB), or both strands (double strand break, DSB). The common cause of this is ionizing radiation. The fourth and last major type of DNA damage is the covalent crosslinkage between bases. This can occur on the same DNA strand (intrastrand) or on opposite strands (interstrand).
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  • There are four primary mechanisms for repairing damage to DNA. The first is direct chemical reversal, often through enzymes. Direct chemical reversal is awfully specific, so the more general repairs are done by excision repair mechanisms. These repairs are classified under base excision repairs (BER), nucleotide rexcision repair (NER), and mismatch repair (MMR).
  • 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 as it shows that each of the various problems require specific mechanisms to fix.
  • Base excision repair has a few steps. First, DNA glycosylases identify and remove damaged bases. Next, its deoxyribose phosphate backbone component is removed, creating a gap. Then, it is replaced with the correct nucleotide, relying on DNA polymerase beta, one of 11+ DNA polymerases encoded by our genes. Finally, the break in the strand is ligated, requiring two ATP reliant enzymes.


DNA ligase repairing chromosomal damage
DNA ligase repairing chromosomal damage


  • 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 proteinf actors. 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 'bad' 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.
  • Mismatch Repair corrects mismatches of normal bases (A&T, C&G). This involves two major steps, the identification of a mismatch and the cutting of the mismatch.
  • Repairing Strand Breaks is necessary after ionizing radiation causes single strand breaks (SSBs) and double strand breaks (DSB) in the backbone. SSB’s in one strand are repaired with the same system of enzymes in BER, whereas DSB arerepaired with two mechanisms. The first is direct joining of the b


Review Questions
  1. Which molecule related to RNAi would be the main player in post-transcription gene silencing in a healthy cell?
    • siRNA
    • tRNA
    • miRNA
    • dsRNA
    • ssRNA
  2. Which of the following is notan example of RNAi
    • Argonaute proteins in a cell that is infected with a virus destroy mRNA made by that virus's RNA.
    • A cell does not transcribe a certain segment of DNA containing a specific gene. The gene is not expressed.
    • When dsRNA for a certain receptor protein is introduced into a cell, those proteins do not appear on the surface of the cell.
    • A cell's miRNA cleaves the mRNA for multiple different genes related to mitochondria activity.
    • The presence of dsRNA in a cell causes an increase in the activity of Dicer proteins. Soon afterwards, the activity of another protein in the cell decreases.
  3. What are the two major types of secondary structure of a protein?
    • Pleated helices and alpha sheets
    • Pleated sheets and alpha helices
    • Beta pleated-sheets and beta helices
    • Helical sheets and alpha helices
    • Alpha pleated sheets and beta helices
  4. What kind of bonds create protein folding?
    • Hydrogen bonding between amino acids
    • Covalent bonding between amino groups and adjacent amino groups
    • Ionic bonding between adjacent carboxyl groups
    • Ionic bonding between amino groups
    • Hydrogen bonding between amino groups and carboxyl groups
  5. Which of the following won’t cause appreciable damage to DNA?
    • Ultraviolet radiation
    • Oxygen radicals
    • Hydrocarbons
    • Infrared radiation
    • Chemotherapy
  6. Which of the following removes an entire nucleotide patch during repair?
    • Direct chemical repair
    • Nucleotide excision repair
    • Base excision repair
    • Mismatch repair
    • Nucleotide removal repair
  7. Which of the following types of DNA damage would result from a proofreading failure during DNA replication?
    • Deamination
    • DNA backbone breakage
    • Base mismatch
    • Single strand break
    • Double strand break
  8. What is the purpose of a modified guanine nucleotide cap?
    • Easier movement
    • Cleave the 3’ end
    • Remove nucleotides
    • Keep RNA from degrading
    • End transcription
  9. What proteins detect branch sites?
    • Introns
    • Extrons
    • SnRNPs
    • RNA polymerase
    • DNA polymerase
  10. What shape do pre-miRNA’s take?
    • Double stranded
    • Hairpin loops
    • Helices
    • Pleated sheets
    • Triple stranded
Essay:
RNAi is thought to be one of the most important recent genetic discoveries.
a. What are two major purposes of RNAi molecules?
b. What are the two major molecules that are used in RNAi?
c. If a cell need to use RNAi to turn off multiple genes that share a common nucleotide sequence, what would it do? Explain the process and the pathway it uses.
d. If a researcher wanted to turn off one gene to examine its function, what would he do? Explain what effect this would have and the pathway it would use.




Sources:
1. The basics of transcription
2. An overview of RNA processing, especially in terms of the cap and tail.
3. A detailed overview of RNA splicing
4. A detailed animation and slideshow about the two main processes of RNAi
5. Some basic facts about RNAi
6. A simplistic video about RNAi and its discovery. Offers very good analogies.
7. Overview of the functions and specifics of RNAi with limited mention of processes.
8. More complicated explanations of purpose of RNAi and methods.