From DNA to RNA The RNA world

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From DNA to RNAThe RNA world
MB 207 Molecular Cell Biology
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TheNucleolus
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Central Dogma of Molecular Biology

• Transcription of DNA to RNA and then to protein
• Represented by 3 major stages.
• The DNA replicates itself Replication
• DNA transcribed to mRNA Transcription
• mRNA carries coded information for protein
  synthesis Translation

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Why would the cell want to have an intermediate
between DNA and the proteins its encodes?

• The DNA can then stay protected, away from the
  caustic chemistry of the cytoplasm.
• Gene information can be amplified by having many
  copies of an RNA made from one copy of DNA.
• Regulation of gene expression can be effected by
  having specific controls at each element of the
  pathway between DNA and proteins.

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Basic structure of RNA

• RNA consist of
• Ribose
• Phosphoric Acid
• Nitrogenous bases Purines (A / G) Pyrimidines
  (C / U)
• The nucleotides are linked together by
  phosphodiester bridges
• RNA is single stranded
• It has extensive regions of complementary AU, or
  GC pairs and the molecule folds on itself forming
  structures called hairpin loops
• Can form various 3-D structure just like proteins

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Types of RNAs
TYPES OF RNA FUNCTION
mRNAs messenger RNAs, code for proteins
rRNAs ribosomal RNAs, form the basic structure of the ribosome and catalyze protein synthesis
tRNAs transfer RNAs, central to protein synthesis as adaptors between mRNA and amino acids
snRNAs small nuclear RNAs, complexed with proteins in the nucleus, involved in RNA splicing
snoRNAs small nucleolar RNAs, used to process and chemically modify rRNAs
Other noncoding RNAs function in diverse cellular processes, including telomere synthesis, X-chromosome inactivation, and the transport of proteins into the ER
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Transcription Procaryotes vs Eucaryotes

• Eucaryotes possess three different types of RNA
  polymerases (I, II, III), instead of one in
  prokaryotes
• Procaryotes polymerase ( only one) contain a s
  factor to initiate transcription without help
  from other proteins
• Eucaryotes required help from large sets of
  proteins called general transcription factors
  before the transcription can be started
• Eucaryotes must deal with DNA that is packed into
  nucleosome and chromatin

Type of polymerase in eukaryotic Genes transcribed
RNA Pol I 5.8S, 18S and 28S rRNA genes
RNA Pol II All protein-coding genes, plus snoRNA genes and some snRNA genes
RNA Pol III tRNA genes, 5S rRNA genes, some snRNA genes and genes for other small RNAs
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DNA Transcription
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Summary of the steps leading from gene to protein
in eukaryotes and bacteria

• Prokaryotes Transcription takes place in
  cytoplasm. When transcription is completed, RNAs
  are ready for use in translation. Translation can
  even begin during transcription.
• Eukaryotic Transcription takes place in nucleus.
  The primary RNA transcripts, are often modified
  in the nucleus before export to the cytoplasm.

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Transcription Initiation involved three defined
steps

• Copying of one strand of DNA into complementary
  RNA sequence by the enzyme RNA polymerase
• Transcription starts at the promoter and proceeds
  in the 5'-to-3' direction (unidirectional)
• A promoter is an oriented DNA sequence that
  points the RNA polymerase in one direction which
  determines which DNA strand is to be copied.
• A promoter is a high-affinity binding site for
  the RNA polymerase. (closed complex)
• Most promoters are at the upstream of where
  transcription will start. DNA is unwinded, base
  pairs are disrupted and produce bubble of
  single stranded DNA. (open complex)
• Promoter consists of consensus sequences
  containing specific strings like TATA (Pribnow
  box) and CAAT
• Transition to the elongation phase (stable
  ternary complex)

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Transcription InitiationConsensus sequences
found in the vicinity of eukaryotic RNA
Polymerase II start points
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Transcription - Initiation

• RNA polymerase required the help of a lot of
  proteins before the transcription can actually
  started
• General transcription factors to recognize the
  promoter and to make specific contact between the
  Polymerase and the DNA
• Transcriptional activators to overcome the
  difficulty of Polymerase and general
  transcription factors binding to the DNA that was
  tightly packaged in chromatin
• Mediators Allow proper communication between
  the activators and the DNA as well as the general
  transcription factors
• Chromatin-modifying enzymes Allow accessibility
  of the whole assemble of transcription initiation
  machinery to the DNA

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Initiation of transcription of a eukaryotic gene
by RNA polymerase II

1. The promoter contains a DNA sequence called the
   TATA box
2. TATA box is recognized and bound by transcription
   factor TFIID, which enables the binding of TFIIB.
3. DNA distortion produced by the binding of TFIID
   is not shown.
4. The rest of the general transcription factors,
   RNA polymerase, assemble at the promotor.
5. TFIIH uses ATP to pry apart the DNA helix at the
   transcription start point, allow transcription to
   begin.

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Transcription Initiation by RNA polymerase II in
a eukaryotic cell
Binds to short sequences in DNA, acting from a
distance (thousands of nt pairs)
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Transcription - Elongation

• The RNA polymerase then stretches open the double
  helix and begins synthesis of an RNA strand
  complementary to one of the strands of DNA
• The strand of DNA from which RNA copies is the
  sense or coding strand
• The other strand, to which its sequence is
  identical to the RNA is the antisense or
  non-coding strand
• Elongation continue with the addition of rNTPs
  (ribonucleic nucleotides triphosphates)
• General transcription factors are released from
  DNA during elongation of RNA transcript.
• Elongation ceased when the enzyme encounters the
  2nd signal in the DNA terminator, where the
  polymerase halts and releases both the DNA and
  the RNA

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Transcription - Elongation

• Steps of elongation
• Unwinding of DNA in front of the enzyme
• Synthesis of RNA
• RNA proofreading (one mismatch consists thousand
  nucleotides)
• a. pyrophosphorolytic editing
• b. hydrolytic editing
• Dissociation of RNA
• Re-annealing of DNA behind the enzyme

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mRNA Processing
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mRNA Processing-transcription elongation in
eukaryotes is tightly coupled to RNA processing

• Capping (5 end)- is the 1st modification of
  eukaryotic pre-mRNAs
• Splicing- removes intron sequences from newly
  transcribed pre-mRNAs
• Polyadenylation (3 terminus) poly-A signal
  sequence

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mRNA Processing

• Eukaryotic mRNAs undergo extensive modifications
  to increase their stability and become
  biologically active.
• Capping
• 5' end of mRNAs is capped with a
  7-methylguanosine triphosphate (7mGTP) shortly
  after initiation (RNA triphosphatase, guanylyl
  transferase and methyl transferase)
• The unique 5' - 5' triphosphate linkage formed
  increase mRNA stability by affording protection
  from exonucleases
• It also brings a recognizable signal for proteins
  involved in subsequent splicing process and also
  during translation
• Allow cells to assess later for an intact mRNA
• Allow cells to differentiate mRNA from other RNAs

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The reaction that cap the 5 end of each RNA
molecule synthesized by RNA polymerase II

• Capping is carried out by 3 enzymes
• Phosphatase
• Guanyl transferase
• Methyl transferase

The structure of the cap at 5 end
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mRNA Splicing

• Eukaryotic genes were broken into small pieces of
  coding sequence (expressed sequences or exons)
  interspersed with long intervening sequences or
  introns
• Both exons and introns are transcribed into RNA
  precursor mRNA
• RNA splicing occur to remove the introns
• Benefits of having exons and introns as well as
  RNA splicing
• Facilitate the emergence of new proteins
• One genes to be spliced in different way to give
  different mRNAs

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Structure of two human genes showing the
arrangement of exons and introns
Alternative splicing of the a-tropomyosin gene
from rat
(regulates contraction in muscle cells)
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• RNA splicing reaction
• The mechanism involves formation of a loop,
  called a lariat, in a process directed by small
  nuclear ribonucleoproteins (snRNPs). The complex
  mRNA-snRNPs is called a spliceosome.

• A specific Adenine nucleotide in the intron
  sequence attacks the 5 splice site
• Cut the sugar phosphate backbone
• Covalently linked 5 end to A nt,
• creating a loop.
• 4. Released free 3OH end of the
• exon sequence, reacts with the
• start of the next exon.
• 5. Joining two exons together, releasing intron
  in the shape of lariat which is subsequently
  degraded.

Creating a loop in the RNA molecule
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RNA splicing

• The consensus nt sequences in an RNA signal the
  beginning and the end of the introns hence
  signal where splicing occurs

YC/U RA/G
For example RNA splicing
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RNA splicing is performed by the spliceosome

• RNA splicing
• Spliceosome is the Splicing machinery
• Consists of 5 short RNA molecules (lt200nt U1,
  U2, U4, U5 U6), known as small nuclear RNAs
  (snRNAs)
• Each of these RNA complexes with at least 7
  protein subunits to form a small nuclear
  ribonucleoproteins (snRNPs)
• Form the core of spliceosome is formed large
  assembly of RNA and protein molecules that
  performs pre-mRNA splicing in cell.

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Polyadenylation of mRNA

• mRNAs are polyadenylated at the 3' end
• Just before termination a specific sequence,
  AAUAAA (polyadenylation site), is recognized by a
  polyadenylate polymerase
• The primary transcript is cleaved approximately
  20 bases downstream and a string of 20 - 250
  adenines termed poly-A tail is added to the 3'
  end

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Transcription - termination

• Terminator trigger the elongation polymerase to
  dissociate from the DNA and release the RNA
  chain.
• Types of bacterial terminators
• Rho-indipendent terminators (intrinsic
  terminators without involvement of other
  factors)
• Rho-dependent terminators (require Rho factor
  to induce termination)

Multiple RNA polymerase can transcribe the same
gene at the same time A cell can synthesize a
large number of RNA transcripts in a short time
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Export of mRNA to the cytoplasm

• After the mRNA been processed special head and
  tail regions are added and some parts are spliced
  out, mRNA leaves the nucleus and carries the code
  into the cytoplasm
• How does the cell distinguish which mRNA is the
  one that is ready to be transported?
• Must be bound by appropriate proteins i.e.
  cap-binding complex
• nuclear ribonucleoproteins (nRNP) involved in RNA
  splicing should be excluded from mature mRNA
• Special feature on mature mRNA i.e. 5 cap and 3
  poly A tail

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Export of mRNA to the cytoplasm

• Nuclear pore complex recognizes and transport
  only completed mRNA
• Aqueous channels in the nuclear membrane
• Connect nucleoplasm and cytosol
• Small molecules (lt 50kDa) can diffuse freely
  through them
• Macromolecules needs to be tagged before they are
  allowed to pass

Transport of a large mRNA molecule through the
nuclear pore complex.
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Summary of transcription
RNA polymerase
-is the primary enzyme of transcription -resembles a crab claw -is generally composed of several subunits -adds nucleotides to the 3end of the growing RNA chain
Elongation Unwinding of DNA in front of the enzyme Synthesis of RNA RNA proofreading Dissociation of RNA Re-annealing of DNA behind the enzyme
Termination Rho-independent terminators Rho-dependent terminators
Transcription cycle
Initiation Formation of a closed complex Transition to an open complex Promoter escape
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Ribosomal RNA (rRNA)

• 80 of RNA in cells (3-5 is mRNA)
• rRNA is the functional product of the rRNA gene,
• Each growing cells require 10 million copies of
  each type of rRNA,
• Each cell contain multiple copies of the rRNA
  genes
• rRNAs form the core of ribosome

• Nucleolus Site of rRNA processing and ribosome
  assembly
• Large aggregate of macromolecules mainly genes
  coded for rRNAs, snoRNAs and proteins required
  for ribosome assembly
• Types of rRNAs
• Eukaryotes 28S, 18S, 5.8S 5S
• Prokaryotes 23S, 16S 5S

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Processing of rRNA
The chemical modification and nucleolytic
processing of an eukaryotic 45S precursor rRNA
molecule into 3 separate ribosomal RNAs

• 28S, 18S, 5.8S are cleaved from a single
  chemically modified large precursor
• Synthesised by RNA Polymerase I at the nucleolus
• No C-terminal tail as compared to RNA Polymerase
  II
• Transcript is not capped nor polyadenylated
  hence rRNAs are retained within nucleus
• Chemical modifications at specific positions
  methylations, isomerization of uridine to
  pseudourine

snoRNAs locate the sites of modification by
base-pairing to complementary sequences on the
precursor rRNA. The snoRNAs are bound to proteins
and the ciplexes are calledsnoRNPs. snoRNPs
contain the RNA modification activities
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• Sites of modification and cleavage of precursor
  to mature rRNAs are by a group of protein bound
  snoRNAs.
• Locate the sites of modification by base-pairing
  to complementary sequences on the precursor rRNA.
• The RNA-protein complexes are called snoRNPs.
• The RNA modification activities, presumably
  contributed by the proteins but possibly by the
  snoRNAs themselves.
• 5S is transcribed by Polymerase III without any
  chemical modification outside the nucleolus.

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Genetic Code

• Sequence of nucleotides in the mRNA is read in
  group of 3, each group of 3 consecutive
  nucleotides in mRNA is called a codon.
• 4 different nucleotides in each position, hence 4
  x 4 x 4 64 combinations of 3 nucleotides
• Only 20 different aa, some aa is specified by
  more than 1 codon (redundancy/degeneracy)
• All 64 codons specifies either 1 aa or a stop to
  the translation process
• Genetic code is generally universal (same btw
  prokaryote eukaryotes) but there are some
  differences in the mitochondria
• Differences in the preference of codon use
  between different species e.g. A giraffe might
  use CGC for arginine much more often than CGA,
  and the reverse might be true for a sperm whale.

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Genetic Code
Reading frame - The phase in which nucleotides
are read in sets of three to encode a protein. A
messenger RNA molecule can be read in any one of
3 reading frames, only one of which will give the
required protein
Start codon bacteria Stard codon eukaryotic
5-AUG-3 5-AUG-3
5-GUG-3
5-UUG-3
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3 possible reading frames in protein synthesis

• Sequence of mRNA is read from 5 to 3 in
  sequential sets of three nucleotides
• The same RNA sequence can specify 3 completely
  different aa sequences, depending on how the
  sequence was read (the reading frame)

• In reality, only one of these reading frames
  contains the actual message
• Special punctuation signal at the beginning of
  each RNA message sets the correct reading frame
  at the start of protein synthesis

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tRNA match amino acids to codons in mRNA

• An adapter that carries a specific aa and
  matches its corresponding codon in mRNA during
  translation
• A mature tRNA, 65 - 95 nts
• Secondary structure cloverleaf
• Tertiary structure - L-like
• Consist of a stem and three main loops
• 3 end of tRNA has a site that attaches to a
  specific aa
• Anticodon loop contain a site with 3 nucleotide
  bases (anticodon) which is complementary to the
  mRNA codon for the aa its carries
• T loop
• D loop

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tRNA

• If there were one tRNA for each codon, there
  would be 64 tRNA types. However, the actual
  number is less than 61
• The reason for this is the versatility of TRNA
  which can bind to more than one codon without
  introducing mistakes
• A single tRNA can recognize the codons for UUU
  and UUC because both code for the same amino
  acid, phenylalanine.
• The flexibility in the pairing between the third
  base of the codon and the corresponding base in
  the anticodon led to proposal of wobble
  hypothesis by Francis Crick.
• Wobble hypothesis postulates that the flexibility
  in codon-anticodon binding allows some unexpected
  base pairs to form eg. inosine

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tRNA

• tRNA is transcribed by RNA polymerase III as a
  large precursor
• tRNA splicing occurs through a cut-and-paste
  mechanism catalysed by proteins
• Post-transcriptional chemical modification alter
  the standard A, U, G C bases
• Aminoacyl-tRNA synthetases couple each amino acid
  to its appropriate set of tRNA molecules

• There are 20 aminoacyl-tRNA synthetases for each
  of the 20 aa
• i) catalyze the attachment of amino acids to
  their corresponding tRNAs via an ester bond.

Amino acid activation