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Title: From DNA to RNA The RNA world


1
From DNA to RNAThe RNA world
MB 207 Molecular Cell Biology
2
TheNucleolus
3
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

4
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.

5
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

6
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
7
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
8
DNA Transcription
9
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.

10
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)

11
Transcription InitiationConsensus sequences
found in the vicinity of eukaryotic RNA
Polymerase II start points
12
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

13
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.

14
Transcription Initiation by RNA polymerase II in
a eukaryotic cell
Binds to short sequences in DNA, acting from a
distance (thousands of nt pairs)
15
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

16
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

17
mRNA Processing
18
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

19
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

20
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
21
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

22
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)
23
  • 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
24
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
25
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.

26
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

27
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
28
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

29
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.
30
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
31
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

32
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
33
  • 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.

34
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.

35
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
36
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

37
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

38
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

39
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
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