Ocular Biochem: Nucleic Acids

What are the two main functions of nucleic acids?

-maintain code for the amino acid sequence for all proteins in a cell

-synthesize those proteins

What are nucleic acids operations needed for?

-growth

-division

-specialized functions

-development and hereditary characteristics

What does uncontrolled cell division cause?

cancer

What are many metabolic diseases caused by?

enzyme defects caused by DNA based hereditary problem

Deoxy Ribonucleic Acid (DNA)

-double stranded, helical polymers of deoxyriboncleotides found in the nucleus of the cell

-function: storage of genetic material

-double stranded in eukaryotic cells having a nucleus

Ribonucleic Acid (RNA)

-single stranded, non-helical polymers of ribonucleotides found in the nucleus and cytoplasm of the cell

-function: to transfer the genetic information from the nucleus to cytoplasm for protein synthesis

Messenger RNA (mRNA)

-a complementary copy of selected regions of the DNA

-carries the genetic message from the nucleus to the cytoplasm and acts as the template for protein synthesis

-made in the nucleus

-used during translation

-comprises only 3% of the total RNA present at a given time, indicates a strict control necessary for the rate of protein synthesis

Ribosomal RNA (rRNA)

-along with proteins, forms the ribosomes which is the site of protein synthesis

-some of them have catalytic and coenzyme functions as well

-represents 75% of all RNA in a cell

-about 66% of the mass of a ribosome where it resides

-may have a catalytic role, but specific role is not understood

Transfer RNA (tRNA or Soluble RNA)

-a short chain RNA that attaches to specific amino acids

-transfer the amino acids from the cytoplasm to the site of protein synthesis

What are the two main types of nucleic acids?

-DNA: nucleic acid form that preserves the code for making all the proteins needed by a cell

-RNA: translates that code into specific proteins

polymers of nitrogen containing hydrophobic bases

What are the hydrophobic bases of DNA?

AGCT

-adenine

-guanine

-cytosine

-thymine

What are the hydrophobic bases of RNA?

AGCU

-adenine

-guanine

-cytosine

-uracil

What are the hydrophobic bases of DNA and RNA joined together by?

hydrophilic pentose phosphates

Nucleoside

-deoxyribose (in DNA) or ribose (in RNA) + base

-prefix "deoxy" used if the sugar is a deoxypentose (2'-hydroxy group is replaced by hydrogen)

Nucleotide

-base + sugar + phosphate, e.g. deoxyadenosine 5'-triphosphate (dATP), cyclic GMP

-dATP can be incorporated into a molecule of DNA

-cyclic GMP: intracellular hormone

What are the bases of DNA like?

-held together by hydrogen bonds

-two bonds between A and T and three bonds between G and C

-are hydrophobic

-deoxypentose phosphates are hydrophilic

-hydrophobic bases held internally in a double helix structure of DNA

Where are deoxypentose phosphates located in DNA?

on the exterior of the helix

What are the different forms that DNA can exist as?

A, B, and Z-form

What do the chains in duplex DNA contain?

-one chain contains the code for the synthesis of specific proteins = coding strand

-other chain contains the template or complement of the code = complementary strand

Genetic Code

-sequence of sets of three bases in the chain

-three bases in succession code for a single amino acid in a protein

How many chromosomes is human cellular DNA divided into in each cell?

46 chromosomes

Metaphase Chromosome

-at metaphase, two chromosomes joined by a centromere

-a centromere is centrally located

-specific DNA sequence is required for cell division

Chromatid

-a single chromsome at metaphase

-composed of super-coiled structures

-super coiled structures have lengths of chromatin fibers

Histone Proteins

assist DNA in compacting itself

Nonhistone Proteins

-bound to the DNA at the strands

-control RNA and RNA processing

DNA Replication

-occurs during the process of cell division

-replication: semiconservative

-one original strand of the parents duplex DNA becomes incorporated into each of the two new daughter duplex DNA molecules

-one coding strand and one complementary strand of each of the two new strands come from the original DNA before division

Where does DNA replication take place?

takes place in several locations at one time since DNA in each chromosome is so long

What does DNA replication require?

-many proteins to carry out replication

-some of these proteins such as DNA polymerase are enzymes

Replication Fork

location of active replication

What is the sequence of synthesis for DNA replication?

-the helix must be unwound before replication

-synthesis of each strand must proceed from the 5' end of each added nucleotide to the 3' end of each growing strand

Leading Strand in DNA Replication

-new strand synthesized in the conventional direction 5'-3'

-each short fragment in the lagging strand is successively formed backwards in the same direction as that of the leading strand

Okazaki Fragments in DNA replication

new strand made in discrete, short discontinuous fragments

What are fragments in DNA replication joined by?

a ligase enzyme to become a continuous strand

What are the enzymes involved in DNA replication?

-DNA helicase

-RNA primase

DNA Helicase

breaks the hydrogen bonds between the bases to form single strands in DNA replication

RNA Primase

-an enzyme bound to DNA helicase in DNA replication

-synthesizes short strands of primer RNA that bind to one of the separated parental strands

What does one of the primer strands in DNA replication become?

-becomes the template for the new lagging strand

-it is crucial that parental strand remains a single strand at this stage

-double-helix-destablizing proteins temporarily bind to the separated strand

RNA Primer

initiator for lagging strand in DNA replication

Where does the parental template for the lagging strand go through during DNA replication?

loops and goes through DNA polymerase III enzyme complex

DNA Polymerase III Enzyme Complex

comprised of two polymerase enzymes:

-lower polymerase III enzyme synthesizes new DNA bound to the RNA primer to form an Okazaki fragment on the lagging strand

-the other polymerase III forms the new DNA chain for the leading strand

DNA Polymerase I and Ligase

-hydrolyze the RNA portion of the primer during DNA replication

-complete the gaps between the DNA Okazaki fragments to form a continuous strand

Ligase Enzyme

binds the last nucleotide to join the fragment with the new growing strand during DNA replication

What makes RNA different from DNA?

-ribose replaces deoxyribose as a pentose

-uracil replaces thymine as a base in most cases

What are some exceptions when RNA is not single stranded?

regions of both transfer and ribosomal RNA as well as some RNA containing viruses

Heterogenous Nuclear RNA (hnRNA)

-initial coding RNA product made from DNA

-synthesized in the nucleus

-contains two or more coding genes (exons) separated by one or more noncoding regions (introns)

-a collection of pre-RNA , nRNA, and ribonucleoproteins involved with pre-RNA

-considered equivalent to pre-RNA

Transcription

-synthesis of RNA from DNA

-DNA serves as the template for new RNA formation in this process

-RNA is made by the catalytic activity of three different RNA polymerases (DNA-directed RNA polymerases)

RNA Polymerase I

for rRNA synthesis

RNA Polymerase II

for hnRNA and ultimately mRNA synthesis

RNA Polymerase III

for tRNA and the smaller species of rRNA

What is the first step in protein synthesis?

-formation of hnRNA

-the polymerization is similar to DNA polymerization (replication)

-nucleotide triphosphate are used as building blocks

What does uridine triphosphate replace during transcription of RNA?

replaces deoxythymidine triphosphate

Where can transcription begin?

only begin at a promoter site on DNA

Promoter Site in Transcription

-helps in rigidly controlling protein synthesis by limiting the amount of RNA that is available

-contains specific short sequences of DNA such as TATA and/or CAAT

-these sequences do not code for any amino acids

What is the role of the promoter site in transcription of RNA?

-numerous regulatory proteins bind to this region

-regulatory proteins support or inhibit the initiation of hnRNA synthesis

-the entire region is referred to as upstream promoter element

-proteins bind to the DNA at their major grooves at linker DNA sites between nucleosomes

What will initiate RNA synthesis during transcription?

-binding of RNA polymerase to the promoter element

-synthesis proceeds downstream on the DNA in a (5'-3') direction

-could stop depending on the combined influences of the proteins gathered at the TATA, CAAT, or other DNA promoting sequence regions

Enhancers

auxillary regions of DNA that have further influence on hnRNA synthesis

What will code for a single amino acid in a protein?

-a sequence of three bases - code is redundant

-more than one code of three bases can signal for the same acid

-three of these codes signal either synthesis initiation or the incorporation of an amino acid

-three stop codes specify only termination of synthesis

Nonsense Sequences

base sequences on both DNA and RNA that do not code for amino acids, start signal, and stop signal

What are nonsense sequences used for?

-spacer molecules, to connect coded regions of gene sequences (introns)

-signal and control transcription (promoters, enhancers)

-attach to microtubules during mitosis and meiosis

-record the number of cell division as an agin marker while preserving chromosome integrity (telomeres)

Exons

gene sequences that code for either all or part of a protein in both DNA and hnRNA

Intron

-noncoding sequence separating exons

-act as spacers

-are eventually looped out of a coding sequence prior to protein synthesis

What does the formation of mRNA begin with?

hnRNA acquiring a cap of GTP at its 5'-end, and tail of 100-200 bases of adenosine phosphate at its 3'-end

What does the GTP cap at the 5'-end of hnRNA and its tail protect during the formation of messenger RNA?

-protects RNA from degradation and involved in initiation of protein synthesis

-tail may help in preventing degradation of RNA

How does mRNA formation from hnRNA occur?

-occurs by looping out the introns and splicing the exons together

-looping out of introns is aided by specialized small nRNA and small nuclear proteins

Transesterification Reactions

-facilitate looping out of introns

-break the intron-exon phosphate bonds

-form a new exon-exon phosphate bond

Where is mRNA transported once it is formed?

transported out of the nucleus

Translation

-protein formation from mRNA

-formation of a polypeptide is a cooperative process between mRNA, tRNA, ribosomes, and specialized proteins

What is the structure of tRNA?

a small RNA molecule consisting of:

-extensive base-pair binding (double helix formation)

-looping

-an amino acid attachment site

-a site for binding to a three base codon of mRNA (anticodon region)

-resembles a human liver due to helical folding

What are the important regions of tRNA that contribute to protein synthesis?

-anticodon region

-3'-end, aka acceptor stem

What are the roles of the bases in tRNA function?

-tRNA uses N, N'-dimethyl guanine as one of the bases

-they facilitate the twisted conformation of tRNA that helps to carry its amino acid to a codon site on a ribosome

What is the process of translation?

-tRNA binds to its amino acid

-tRNA bound to amino acid enters the ribosome to attach to the codon portion of mRNA with its anticodon site

-ribosome itself is a two part assembly with each part composed of both proteins and rRNA

Smaller Subunit 40S

houses the mRNA during translation

Larger Subunit 60S

houses the incoming tRNAs and their amino acids during translation

What occurs during protein synthesis (translation)?

-one or more ribosomes move along the mRNA chain and simultaneously synthesize a polypeptide

-each tRNA with its amino acid binds to mRNA in succession

-each amino acid coming into the 60S subunit forms a peptide bond with the previous amino acid and the peptide chain lengthens

Peptidyl Transferase Activity

-peptide bond is formed by the catalytic activity of the 23S rRNA located on the ribosome

-ribosome continues to move along the mRNA chain (peptide bond formation) until all of the code is read

How many proteins can be formed from one mRNA molecule per minute?

10 proteins

How many proteins are needed for the initiation of the translation process in eukaryotes?

12 associated proteins

What are the five stages of protein synthesis?

-initiation

-elongation 1

-elongation 2

-elongation 3

-termination

What is the initiation stage of protein synthesis?

-ribosome is assembled in response to its binding to mRNA and the first tRNA

-the tRNA(Met) binds to the P-site (peptidyl) on the 40S ribosome where the codon for mRNA reads AUG

-in response to this activity, the 60S ribosome binds to the 40S ribosome

-several initiation factors (proteins) aid in this process

What does GTP act as during the initiation stage of protein synthesis?

an energy source for the process to occur

What is Elongation Stage 1 of protein synthesis?

-a second tRNA delivers the proper amino acid to the A-site (aminoacyl)

-this occurs adjacent to the P-site with identification and binding to the second codon on the mRNA

-GTP is used as an energy source

What is Elongation Stage II of protein synthesis?

-peptide bond formation takes place

-accomplished by the catalytic activity of the 23S rRNA (aka ribozyme) located in the 60S ribosome

-upon completion of peptide bond formation, the amino acid in the P-site is released from its tRNA on the P-site

What is Elongation Stage III of protein synthesis?

-initial tRNA is released from its codon as the ribosome moves along the mRNA

-the dipeptide is transferred from the A-site to the P-site using GTP to supply energy for the process

-elongation is repeated as long as there is more code to be read from mRNA

What occurs during the termination stage of protein synthesis?

-a releasing factor binds to the termination code on mRNA at the ribosomal A-site

-completed polypeptide is released from the ribosome

-ribosome components are also released from the mRNA

-some protein synthesizing ribosomes exist independently in the cytoplasm

-other ribosomes attach themselves to the rough ER

What are the ribosomes that attach themselves to the rough ER after the termination stage of protein synthesis used for?

they will make proteins used for cell membranes, lysosomes, and for extracellular functions

Is the mechanism of protein synthesis identical in both ocular and nonocular cells?

YES

Modification of DNA

a profound influence upon the organism

Sickle Cell Anemia

-occurs due to a genetic code change in DNA

-substitution of valine for glutamine in hemoglobin

-red blood cells develop a sickle-cell shape - blocked vessels and early destruction of the RBCs

How does DNA prevent disease development?

-DNA copies itself faithfully to prevent disease development

-it will enact reparative mechanisms whenever damaged by outside chemical or physical forces

How is DNA damaged by UV radiation?

-UV will induce a bond to form between adjacent pyrimidines in DNA

-these pyrimidines are located side by side in the same DNA chain

-DNA helix is distorted and replication cannot occur beyond the dimer

-a cell having a significant amount of DNA damage may either die or mutate into a cancer cell (skin cancer)

DNA Repair Mechanism

-a variety of repair mechanisms

-prevents cells from undergoing death or becoming cancerous

-a common mechanism requires sequential activity of 3 enzymes: exinuclease, DNA polymerase I, and DNA ligase

Exinuclease

breaks 5'-3' bonds approximately 5 to 8 bases on both sides of the pyrimidine dimer

DNA Polymerase I

-acts following removal of damaged sequence

-sequences new bases at the gap using the opposite chain as a template

DNA Ligase

adds the last base to rejoin the two segments of DNA together again during the DNA repair mechanism

What is the DNA repair mechanism of the 3 enzymes referred to as?

cut, patch, and splice mechanism, it operates continuously to keep DNA intact

Xeroderma Pigmentosum

-repair mechanisms are defective

-pre-existing genetic defect makes imperfect exinuclease molecules

-these molecules are unable to remove pyrimidine dimers efficiently

-patients with this disease develop skin cancer very easily

What are the different responses by the cornea to UV?

-low levels of exposure: an inhibition of cellular mitosis

-moderate to high levels: swollen nuclei and cell death

-extreme levels: complete sloughing of epithelial cells; at higher levels the process of damage is assisted by oxygen

Where is UV damage confined to below 290 nm?

corneal epithelium

Where is UV damage confined to at higher wavelengths (>290 nm)?

damaging effects penetrate into deeper layers of the cornea lens and the retina

How can UV-B radiation (180-320 nm) cause damage to the lens?

-induces DNA damage to lens epithelium

-linked with the formation of cortical and posterior subcapsular cataracts

x-rays and gamma-rays produce even greater damage, they possess higher energy content