Leu, Trp 1/2 life 3 minutes Cys, Ala, Ser, Thr, Gly

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Fundamentals of Nucleic Acid Biochemistry RNA

• Donna Sullivan, PhD
• Division of Infectious Diseases
• University of Mississippi Medical Center

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DNA AND RNA BASIC STRUCTURE
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Repeating Nucleotide Subunits In DNA and RNA
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STRUCTURE OF RNA

• SUGAR
• Ribose
• Phosphate group
• Nitrogen containing base
• Adenine
• Guanine
• Cytosine
• Uracil

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THE NUCLEOTIDE RNA
OH OP-O-5CH2
BASE OH O
4C 1C H
H H H
3C 2C
OH 0H

Adenine Guanine Cytosine Uracil
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The Forms Of RNA Not Just Another Helix

• Does not normally exist as a double helix,
  although it can under some conditions
• Can have secondary structure
• Hairpins pairing of bases within 5-10 nt
• Stem-loops pairing of bases separated by gt50 nt
• Can have tertiary structure
• Pseudoknot, cloverleaf

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RNA Structures
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Structure Of RNA
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Structure of tRNA
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Structure of rRNA
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RNA Replication vs. DNA Replication

• RNA replication
• Requires no priming
• Has many more initiation sites
• Is slower (50100 b/sec vs. 1000 b/sec)
• Has lower fidelity
• Is more processive

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Transcription

• Transcription is the enzyme-dependent process of
  generating RNA from DNA.
• The process is catalyzed by a DNA-dependent RNA
  polymerase enzyme.
• Only coding segments of DNA (genes) are
  transcribed.
• Types of genes include structural genes (encode
  protein), transfer RNA (tRNA), and ribosomal RNA
  (rRNA).

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Transcription

• Transition from DNA to RNA
• Initiation Gene recognition
• RNA polymerase enzyme and DNA form a stable
  complex at the gene promoter.
• Promoter Specific DNA sequence that acts as a
  transcription start site.
• Synthesis of RNA proceeds using DNA as a
  template.
• Only one strand (coding strand) is transcribed,
  the other strand has structural function.
• Transcription factors are proteins that function
  in combination to recognize and regulate
  transcription of different genes.
• Termination signal

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RNA POLYMERASES

• Cellular RNA polymerase
• RNA pol I transcribes rRNA genes
• RNA pol II transcribes protein encoding genes
• RNA pol III transcribes tRNA genes
• Viral RNA polymerases
• Reverse transcriptase of retroviruses
• RNA dependent RNA polymerases of negative
  stranded viruses

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RNA Polymerase Enzymes(DNA-dependent RNA
Polymerase)

• RNA Polymerase I transcribes most rRNA genes (RNA
  component of ribosomes).
• RNA Polymerase II transcribes structural genes
  that encode protein.
• RNA Polymerase III transcribes tRNA genes (for
  transfer RNAs).
• RNA Polymerase IV is the mitochondrial RNA
  polymerase enzyme.

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RNA TRANSCRIPTION
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General Organization of a Eukaryotic Gene
Promoter/Enhancer
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Gene Structure
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Nuclear Processing of RNA

• Chemical modification reactions (addition of the
  5 CAP)
• Splicing reactions (removal of intronic
  sequences)
• Polyadenylation (addition of the 3 polyA tail)

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Processing of mRNA

• In eukaryotes, the mRNA molecule that is released
  after transcription is called precursor mRNA or
  pre-mRNA.
• It undergoes several changes before being
  exported out of the nucleus as mRNA.

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Processing of mRNA

• 5 end is capped with a modified form of the G
  nucleotide known as the 5 cap
• At the 3 end, an enzyme adds a long series of A
  nucleotides referred to as a poly-A tail
• It serves to protect the mRNA from enzymes in the
  cytoplasm that may break it down
• The greater the length of the poly-a tail, the
  more stable the mRNA molecule

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RNA Transcription and Processing

• The process of RNA transcription results in the
  generation of a primary RNA transcript (hnRNA)
  that contains both exons (coding segments) and
  introns (noncoding segments).
• The noncoding sequences must be removed from the
  primary RNA transcript during RNA processing to
  generate a mature mRNA transcript that can be
  properly translated into a protein product.

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RNA Processing

• Capping of the 5-end of mRNA is required for
  efficient translation of the transcript (special
  nucleotide structure).
• Polyadenylation at the 3-end of mRNA is thought
  to contribute to mRNA stability (PolyA tail).
• Once processed, the mature mRNA exits the nucleus
  and enters the cytoplasm where translation takes
  place.

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RNA ProcessingBiogenesis of Mature mRNA
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RNA ProcessingFundamentals of RNA Splicing
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RNA Processing Spliceosomes

• Ribonucleoproteins (snRNPs) function in RNA
  processing to remove intronic sequences from the
  primary RNA transcript (intron splicing).
• Alternative splicing allows for the generation of
  different mRNAs from the same primary RNA
  transcript by cutting and joining the RNA strand
  at different locations.
• snRNPs are composed of small RNA molecules and
  several protein molecules.
• Five subunits form the functional spliceosome.

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RNA ProcessingAlternative Splicing of RNA
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Structural Genes Encode Proteins

• The majority of structural genes in the human
  genome are much larger than necessary to encode
  their protein product.
• Structural genes are composed of coding and
  noncoding segments of DNA.

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Structural Genes Encode Proteins

• The structure of a typical human gene includes
  informational sequences (coding segments termed
  exons) interrupted by noncoding segments of DNA
  (termed introns).
• The exon-introncontaining regions of genes are
  flanked by non transcribed segments of DNA that
  contribute to gene regulation.

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Factors That Control Gene Expression Include

• Cis elements are DNAsequences.
• Trans elementsare proteins.

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Control of Gene Expression

• Primary level of control is regulation of gene
  transcription activity.
• TATA box contained within the gene promoter
  provides binding sites for RNA polymerase.
• Enhancer sequences can be sited very far away
  from the gene promoter and provide for
  tissue-specific patterns of gene expression.

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Gene Enhancer Sequences
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Protein binding sequences in the Promoter region
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Gene Regulation Two types of regulation

• Negative regulation
• Substrate induction (lac operon) gene OFF unless
  substrate is present
• End product repression (trp operon) gene OFF if
  end product is present
• Common in bacteria
• Positive regulation
• Gene is OFF until a protein turns it ON
• Regulatory proteins turn gene ON
• Occurs in eukaryotes

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Negative Regulation
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Positive Regulation
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Lac Operon

• Operon
• Gene organization in bacteria in which several
  proteins are coded by one mRNA
• Allows all proteins to be controlled together

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Differences Between Prokaryotes And Eukaryotes

• Prokaryote gene expression typically is regulated
  by an operon, the collection of controlling sites
  adjacent to protein-coding sequence.
• Eukaryotic genes also are regulated in units of
  protein-coding sequences and adjacent controlling
  sites, but operons are not known to occur.
• Eukaryotic gene regulation is more complex
  because eukaryotes possess a nucleus
  (transcription and translation are not coupled).

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Two Categories Of Eukaryotic Gene Regulation

• Short-term - genes are quickly turned on or off
  in response to the environment and demands of the
  cell.
• Long-term - genes for development and
  differentiation.

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Eukaryote Gene Expression Is Regulated At Six
Levels

• Transcription
• RNA processing
• mRNA transport
• mRNA translation
• mRNA degradation
• Protein degradation

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Transcription Control Of Gene Regulation
Controlled By Promoters

• Occur upstream of the transcription start site.
• Some determine where transcription begins (e.g.,
  TATA), whereas others determine if transcription
  begins.
• Promoters are activated by highly specialized
  transcription factor (TF) proteins (specific TFs
  bind specific promoters).
• One or many promoters (each with specific TF
  proteins) may occur for any given gene.
• Promoters may be positively or negatively
  regulated.

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Transcription Control Of Gene Regulation
Controlled By Enhancers

• Occur upstream or downstream of the transcription
  start site.
• Regulatory proteins bind specific enhancer
  sequences binding is determined by the DNA
  sequence.
• Loops may form in DNA bound to TFs and make
  contact with enhancer elements.
• Interactions of regulatory proteins determine if
  transcription is activated or repressed
  (positively or negatively regulated).

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Chromosome Structure, Eukaryote Chromosomes, And
Histones

• Prokaryotes lack histones and other structural
  proteins, so access to the DNA is
  straightforward.
• Eukaryotes possess histones, and histones repress
  transcription because they interfere with
  proteins that bind to DNA.
• Verified by DNase I sensitivity experiments
• DNase I readily degrades transcriptionally active
  DNA.
• Histones shield non-transcribed DNA from DNase I,
  and DNA does not degrade as readily.

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Chromosome Structure, Eukaryote Chromosomes, And
Histones

• If you experimentally add histones and promoter
  binding proteins histones competitively bind to
  promoters and inhibit transcription.
• Solution transcriptionally active genes possess
  looser chromosome structures than inactive genes.
• Histones are acetylated and phosphorylated,
  altering their ability to bind to DNA.
• Enhancer binding proteins competitively block
  histones if they are added experimentally with
  histones and promoter-binding TFs.
• RNA polymerase and TFs step-around the
  histones/nucloesome and transcription occurs.

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Genomic Imprinting(Silencing)

• Methylation of DNA inhibits transcription of some
  genes.
• Methylation usually occurs on cytosines or
  adenines.
• 5-methyl cytosine
• N-6 methyl adenine
• N-4 methyl cytosine
• CpG islands are sites of methylation in human
  DNA.
• CpG Island

ggaggagcgcgcggcggcggccagagaaaaa gccgcagcggcgcgcg
cgcacccggacagccgg cggaggcggg...
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DNA Methylation And Transcription Control

• Small percentages of newly synthesized DNAs (3
  in mammals) are chemically modified by
  methylation.
• Methylation occurs most often in symmetrical CG
  sequences.
• Transcriptionally active genes possess
  significantly lower levels of methylated DNA than
  inactive genes.
• A gene for methylation is essential for
  development in mice (turning off a gene also can
  be important).
• Methylation results in a human disease called
  fragile X syndrome FMR-1 gene is silenced by
  methylation.

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Hormone Regulation Example Of Short-term
Regulation Of Transcription

• Cells of higher eukaryotes are specialized and
  generally shielded from rapid changes in the
  external environment.
• Hormone signals are one mechanism for regulating
  transcription in response to demands of the
  environment.
• Hormones act as inducers produced by one cell and
  cause a physiological response in another cell.

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Hormone Regulation Example Of Short-term
Regulation Of Transcription

• Hormones act only on target cells with hormone
  specific receptors, and levels of hormones are
  maintained by feedback pathways.
• Hormones deliver signals in two different ways
• Steroid hormones pass through the cell membrane
  and bind cytoplasmic receptors, which together
  bind directly to DNA and regulate gene
  expression.
• Polypeptide hormones bind at the cell surface and
  activate transmembrane enzymes to produce second
  messengers (such as cAMP) that activate gene
  transcription.

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Examples Of Mammalian Steroid Hormones
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Hormone Regulation

• Genes regulated by steroid hormones possess
  binding regions in the sequence called steroid
  hormone response elements (HREs).
• HREs often occur in multiple copies in enhancer
  sequence regions.
• When steroid is absent Receptor is bound and
  guarded by chaperone proteins transcription
  does not occur.
• When steroid is present Steroid displaces the
  chaperone protein, binds the receptor, and binds
  the HRE sequence transcription begins.

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Model Of Glucocorticoid Steroid Hormone Regulation
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RNA Processing Control

• RNA processing regulates mRNA production from
  precursor RNAs.
• Two independent regulatory mechanisms occur
• Alternative polyadenylation where the polyA
  tail is added
• Alternative splicing which exons are spliced
• Alternative polyadenylation and splicing can
  occur together.
• Examples
• Human calcitonin (CALC) gene in thyroid and
  neuronal cells
• Sex determination in Drosophila

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Alternative Polyadenylation And Splicing Of The
Human CACL Gene In Thyroid And Neuronal Cells
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mRNA Transport Control

• Eukaryote mRNA transport is regulated.
• Some experiments show 1/2 of primary transcripts
  never leave the nucleus and are degraded.
• Mature mRNAs exit through the nuclear pores.

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mRNA Transport Control

• Unfertilized eggs are an example, in which mRNAs
  (stored in the egg/no new mRNA synthesis) show
  increased translation after fertilization).
• Stored mRNAs are protected by proteins that
  inhibit translation.
• Poly(A) tails promote translation.
• Stored mRNAs usually have short poly(A) tails
  (15-90 As vs 100-300 As).
• Specific mRNAs are marked for deadenylation
  (tail-chopping) prior to storage by AU-rich
  sequences in 3-UTR.
• Activation occurs when an enzyme recognizes
  AU-rich element and adds 150 As to create a full
  length poly(A) tail.

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mRNA Degradation Control

• All RNAs in the cytoplasm are subject to
  degradation.
• tRNAs and rRNAs usually are very stable mRNAs
  vary considerably (minutes to months).
• Stability may change in response to regulatory
  signals and is thought to be a major regulatory
  control point.
• Various sequences and processes affect mRNA
  half-life
• AU-rich elements
• Secondary structure
• Deadenylation enzymes remove As from poly(A) tail
• 5 de-capping
• Internal cleavage of mRNA and fragment degradation

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Post-translational Control - Protein Degradation

• Proteins can be short-lived (e.g., steroid
  receptors) or long-lived (e.g., lens proteins in
  your eyes).
• Protein degradation in eukaryotes requires a
  protein co-factor called ubiquitin.
• Ubiquitin binds to proteins and identifies them
  for degradation by proteolytic enzymes.
• Amino acid at the N-terminus is correlated with
  protein stability and determines rate of
  ubiquitin binding.
• Arg, Lys, Phe, Leu, Trp 1/2 life 3 minutes
• Cys, Ala, Ser, Thr, Gly, Val, Pro, Met 1/2 life
  20 hours

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Epigenetics

• Non-sequence specific, heritable traits
• Transcriptional gene silencing (TGS)
• Imprinting
• X-inactivation
• RNA-induced transcriptional silencing (RITS)
• Post-transcriptional gene silencing (PTGS)
• RNA-induced silencing complex (RISC)
• G quartets
• Post-translational protein-protein interactions

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RNAi

• First described in C. elegans
• Injecting antisense RNA into oocytes (ssRNA that
  is complementary to mRNA)
• Silences gene expression
• Also injected dsRNA into oocytes found it was 10X
  more potent in inhibiting expression
• Term RNAi

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RNAi

• Higher eukaryotes produce a class of small RNAs
  that mediate silencing of some genes
• Small RNAs interact with mRNA in the 3UTR and
  this results in either mRNA degradation or
  translation inhibition
• Controls developmental timing in at least some
  organisms
• Used as a mechanism to protect against invading
  RNA viruses (plants)
• Controls the activity of transposons
• Formation of heterochromatin

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Mechanism of RNA Interference
Step 1
Step 2
Nat Rev Genet. 2002 Oct3(10)737-47. Review.
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Epigenetics

• The study of mechanisms by which genes bring
  about their phenotypic effects
• Epigenetic changes influence Phenotype without
  altering Genotype
• Changes in properties of a cell that are
  inherited but dont represent a change in genetic
  information.

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A Histone Code?

• Modifications of histones (usually the amino
  terminal tails) convey epigenetic information
• Types of modifications
• Acetylation and methylation of lysine.
• Methylation of arginine
• Phosphorylation of serine
• Ubiquitination of lysine.
• The histone code hypothesis posits that serial
  modifications provide a blueprint for reading
  chromatin -for transcription, replication,
  repair, and recombination.

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Remodeling Nucleosomes

• Changing the way nucleosomes bind to the DNA in
  chromosomes is important to allow access to the
  underlying DNA sequences during DNA replication,
  repair, recombination, and transcription.
• This occurs in three general ways
• Modification of the lys and Arg residues on the
  histone tails decreases the grip of the
  nucleosome on DNA and causes the nucleosome to
  slide more easily.
• Variant histones are added to pre-existing
  nucleosomes
• ATP-dependent protein remodelingcomplexes cause
  nucleosomes to dissociate and/or slide along the
  DNA.