DNA and RNA Functions and Structure

• Nucleic acids include DNA and RNA

DNA Function

• DNA: Deoxyribonucleic acid, carries genetic instructions for development, functioning, and reproduction in living organisms and some viruses

RNA Function

• RNA: Ribonucleic acid, can carry genetic instructions in viruses
• 3 RNA types convert DNA code into polypeptides (proteins)
• RNA can act as a catalyst like enzymes
• RNA has regulatory roles in cells, like gene expression and modifying other RNA

Nucleic Acid Structure

• Monomer is a nucleotide consisting of:
  • Sugar
  • Nitrogenous base
  • Phosphate group
• Nitrogen-containing bases project from the backbone and link to a carbon in the sugar with a glycosidic bond
• DNA, a huge polymer, has 249 million base pairs in human chromosome 1

The Sugars

• 5-carbon sugars are present
• DNA has deoxyribose
• RNA has ribose
• Phosphate groups at the 5' carbon bond to the 3' carbon of the next sugar
• Bonds to the nitrogenous base at the 1' carbon

DNA Strands

• DNA strands are antiparallel and run in opposite directions
• One strand runs 5' to 3', while the other runs 3' to 5'
• There are 5 sugar bonds to phosphate
• There is a 3' hydroxyl group on the sugar
• DNA is most stable when double-stranded and twisted into a 3D helix

DNA Packaging

• In eukaryotes, DNA wraps around proteins called histones
• This coiled form = chromatin
• Chromatin further compresses through supercoiling, creating highly compacted structures (chromosomes)
• Most prokaryotes lack histones but have supercoiled DNA forms held together by special proteins

DNA Replication

• 3 key processes:
  • Separating DNA
  • Building a complementary strand
  • Quality control and DNA repair

Separating DNA

• DNA helicase (enzyme) unzips DNA
• Helicase breaks hydrogen bonds between complementary base pairs
• The junction = replication fork

Problem and Solution

• Single-stranded binding proteins (SSBs) prevent hydrogen bonding between base pairs by binding to exposed DNA single strands
• Topoisomerase regulates DNA unwinding

Building Complementary Strands

• DNA polymerase pairs complementary bases to separated strands (template strands) to create a new strand
• DNA polymerase travels in the 3' to 5' direction on the template strand, building the complementary strand in the 5' to 3' direction
• DNA polymerase cannot initiate a strand, so primase (another enzyme) adds a short segment of nucleotides called a primer (short RNA sequence) to the 3' end of the template strand
• DNA polymerase removes primers and replaces them with DNA
• Primase is part of the RNA polymerase enzyme family

DNA Polymerase

• It Is a family of enzymes involved in DNA replication, with 7 different subgroups
• DNA polymerases I, II, III are specific enzymes from E. coli, where DNA replication was first studied

Leading Strand

• The leading strand follows the 3' to 5' template strand by moving toward the replication fork and replicating continuously

Lagging Strand

• The lagging strand moves from the replication fork to the 5' end of the template strand, so replication is discontinuous
• The resulting fragments are Okazaki fragments
• DNA ligase catalyses the reaction to connect fragments
• Replication is semi-conservative because one half of the original strand is conserved

Genetic Code

• Gene: A DNA sequence that codes for a protein
• Protein-coding genes account for less than 2% of our genome's nucleotides
• Each 3-nucleotide base pair codes for an amino acid = codon
• Other nucleotide portions provide information, including protein production controls

Universal Genetic Code

• Almost all protein-coding regions begin with AUG sequence(which encodes methionine)
• There are 3 "stop" codons that mark the end of the protein-coding region
• Multiple codons can code for the same amino acid

Central Dogma

• Transcription: Copying information in DNA to messenger RNA (mRNA)
• Translation: Ribosomes read mRNA and assemble amino acids into a sequence (protein or polypeptide)

Additional Information

• mRNA (messenger): transcription to translation
• tRNA carries corresponding amino acids to the codon and anticodon, which line up to release amino acids

Transcription from DNA to RNA

• Only one DNA strand serves as a template for transcription at a given time
• The template strand of DNA is referred to as the non-coding strand
• The non-template strand is referred to as the coding strand because its sequence will be the same as the new messenger RNA (mRNA molecule)

The 3 Stages of Transcription

• Initiation: RNA polymerase binds to template DNA
• Elongation: RNA polymerase reads DNA and adds nucleotides to mRNA's 3' end (building from 5' to 3')
• Termination: RNA synthesis ceases and RNA polymerase releases mRNA
• Transcription is less complex in prokaryotes than eukaryotes

Eukaryotic Transcription

• Initiation: RNA polymerase attaches upstream of a gene at a specialized sequence called a promoter

  • The promoter region usually has a string of Ts and As, and the DNA helix begins to unwind

• Elongation: the double helix unwinds as RNA polymerase reads the template strand

  • Nucleotides are added to the mRNA at the 3’ end, and the transcribed DNA reforms the double helix

• Termination: RNA polymerase reaches a terminator sequence in the gene

• mRNA and RNA polymerase are released from the new region, and RNA Polymerase can read another gene

• The mRNA will travel to a ribosome

Modification Post Transcription

• A 5' cap is added and will play a role in ribosome initiation
• A Poly-A tail is added to protect it from enzymes in the cytoplasm
• A pre-mRNA consists of exons (segments that code for a protein) and introns (non-coding regions)
• Introns are removed, while exons are joined together by spliceosomes (RNA and protein complexes)

Alternate Splicing

• Exons are joined together in different combinations
• Allows for the production of different mRNAs
• The mRNA is then translated to produce related proteins

Translation & Protein Synthesis

• Translation the process in which a protein is synthesized from the information in its mRNA
• Translation occurs in three main stages:
  • Initiation
  • Elongation
  • Termination
• Ribosomes
  • Ribosomes are organelles that are comprised of both protein and rRNA
  • Translation occurs inside of ribosomes

Transfer RNA (tRNA)

• A small, single stranded RNA
• Contains an anticodon with a sequence of 3 bases that are complementary to the mRNA sequence being read
• Initiation: Translation begins when a small ribosomal subunit attaches to the 5’ cap end of mRNA and travels along towards the mRNA initiation site
  • Next, tRNA with an anticodon binds to the start codon of mRNA - AUG for methionine
  • A large ribosomal subunit then binds and forms the E (exit), P (peptidyl), and A (aminoacyl) sites within the ribosome

Elongation

• The first tRNA occupies the P site
• tRNA enters the A-site
• The amino acids from the P-site then bind to the amino acids at at the a-site
• The ribosome moves along
• The first tRNA exits, and the tRNA moves to the p-site
• A new tRNA enters the a-site, and the process continues as the chain builds a polypeptide strand
• tRNA at the e-site with no amino acid is released

Termination of Translation

• Termination: When a stop codon is encountered, a release factor enters the A site
  • This signals for translation to terminate
  • The ribosome then dissociates, and the newly formed polypeptide is released

From Polypeptide to Protein

• After creation of the polypeptide strand, several changes must occur to process it from a polypeptide into a protein
  • The polypeptide must be folded appropriately in a 3-dimensional shape
  • If it is a quaternary protein, multiple polypeptides need to be assembled

Gene Regulation

• Not all genes need to be on all the time because cells must turn some on and some off
• Volume must be adjusted for each gene and also ensure proteins are properly produced

Regulation in Prokaryotes Compared to Eukaryotes

• Prokaryotes- use a feedback model called the operon system
  • Operon refers to a cluster of coregulated genes that share a promoter and operator
• Eukaryotes- is more complex than in prokaryotes

The Operon System

• The promoter is the site where RNA transcription occurs
• The operator is sequence of bases that is able to control the transcription that allows the repressor to bind and halts gene transcription
• Structural genes are the genes coding for the protein(s) which, when transcribed act as a unit

Lac Operon

• lac operon Produces 3 proteins
• β-galactosidase- splits the bond in lactose
• galactoside permease: a transport protein that embeds in cell membranes and then pumps lactose into the cell
• transacetylase: transfers an acetyl group from one molecule to another
• Lac repressor is always present, so genes are normally off

Lac Repressor

• The repressor is bound to the operator region, and thus the DNA Polymerase cannot transcribe DNA
• Lactose (inducer) then binds to the repressor protein, and then RNA polymerase reads the DNA, as mRNA is transcribed for proteins needed for lactose metabolism
• Once the lactose levels rise, the repressor binds so an upper region and shuts down production stops making the protein

TRP Operon

• These genes are normally on and can produce tryptophan
• If Tryptophan is present, it binds to the repressor and prevents more Tryptophan from being built

Eukaryotic Gene Regulation

• There are four classifications related to what stage of synthesis is being regulated
  • Transcriptional Regulation (Copying mRNA)
    • Regulates which genes are transcribed or the rate of how they are copied;
    • May involve controlling for which genes are transcribed or altering the rate of transcription
    • Access to promoters is either enhanced or decreased
  • Post Transcriptional (After Copying MRNA)
    • Controls the availability of transcribed mRNA to ribosomes
    • Example: Alternative Splicing Perhaps 75% of human genes undergo this
  • Translation Regulation (Protein is being Synthesized) - Controls: How often and how mRNA is translated to a protein - Variation of mRNA length as well
  • Post translational (After Protein is Synthesized)
    • Controls when proteins become functional, how long they are functional, and when they are degraded
    • Example: activation of P53, a tumor suppressor protein by phosphorylation

Mutations

• Mutations are changes in the DNA
• There are two general categories of mutations:
  • Small scale
  • Large scale

Small Scale Mutations

• Include two variations Point mutations- refer to singular base changes Changes of small group pairs

Types of Mutations

• Include:
  • Substituition: The replacing of one base by another
  • Insertion: Placement of a nucleotide within a sequence
  • Deletion is where a singular nucleotide within a sequence is removed
  • Inversion refers to the swapping of two adjacent bases within the sequence -Note: Deletion and Insertion can cause alterations within the reading frame of codons

Effects of Mutations

Effects on Protein Production if mutations take place:

• Silent. This has dues not change the Amino acid coded because of the degeneracy of the code.
• Nonsense: converts a codon into a stop signal
• Missense results in a substitution of an animo acid

Frame Shift Mutation

Insertion or deletion of one or more base pairs causes the reading frame to shift in one direction or the other. The result is multiple missense and/or the occurrence of nonsense mutations.

Large Scale Mutations

Large parts will include large parts of the DNA within it being inserted, lost, repeated, duplicated, or missing all in conjunction with one another. It may include:

• Large chunks of DNA that are inserted, lost, or repeated.
• Duplication or loss of genes.
• Whole regions of chromosomes.

Causes of Mutations

• Mutations during Spontaneous occur during DNA copying, and are the result of damages done to damages to bases, not caused by external factors
• Induced mutations are damage caused to an organism due to its exposure to mutagenic agents, such as with exposure being done through UV and x-ray radiation or even dangerous chemicals

Example Alteration: Sickle Cell Anemia

• The alteration of single nucleotide results in what is now The Sixth amino acid is now being valine instead of glutamic
• The changes of the shapes the proteins of hemoglobin makes now causes them to be rigid rods that can stick and intercept normal blood flow

Additional Information on Mutations

• Mutations: Provides material for genetic diversity thus, they are essential for evolution
  • Can be beneficial, harmful, or neutral.
  • The rates of mutation may be altered because through its interaction of its external environment