from dna to rna the rna world
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MB 207 – Molecular Cell Biology. From DNA to RNA The RNA world. The Nucleolus. Central Dogma of Molecular Biology. Transcription of DNA to RNA and then to protein Represented by 3 major stages. The DNA replicates itself: R eplication DNA transcribed to mRNA: T ranscription

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central dogma of molecular biology
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
why would the cell want to have an intermediate between dna and the proteins its encodes
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.
basic structure of rna
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
transcription procaryotes vs eucaryotes
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 σ 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
slide9

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.

Summary of the steps leading from gene to protein in eukaryotes and bacteria

transcription initiation involved three defined steps
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 promoterand 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)
slide11
Transcription – InitiationConsensus sequences found in the vicinity of eukaryotic RNA Polymerase II start points
transcription initiation
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
slide13

Initiation of transcription of a eukaryotic gene by RNA polymerase II

  • The promoter contains a DNA sequence called the TATA box
  • TATA box is recognized and bound by transcription factor TFIID, which enables the binding of TFIIB.
  • DNA distortion produced by the binding of TFIID is not shown.
  • The rest of the general transcription factors, RNA polymerase, assemble at the promotor.
  • TFIIH uses ATP to pry apart the DNA helix at the transcription start point, allow transcription to begin.
transcription initiation by rna polymerase ii in a eukaryotic cell
Transcription Initiation by RNA polymerase II in a eukaryotic cell

Binds to short sequences in DNA,

acting from a distance (thousands of nt pairs)

transcription elongation
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 it’s sequence is identical to the RNA is the antisenseor 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
slide16

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
mrna processing transcription elongation in eukaryotes is tightly coupled to rna processing
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
mrna processing1
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
slide20

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

mrna splicing
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
slide22

Structure of two human genes showing the arrangement of exonsand introns

Alternative splicing of the a-tropomyosin gene from rat

(regulates contraction in muscle cells)

slide23

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 3’OH 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.
  • 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.

Creating a loop in the RNA molecule

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

Y=C/U

R=A/G

For example: RNA splicing

rna splicing is performed by the spliceosome
RNA splicing is performed by the spliceosome
  • RNA splicing:
    • Spliceosomeis the Splicing machinery
      • Consists of 5 short RNA molecules (<200nt; 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.
polyadenylation of mrna
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
slide27

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

export of mrna to the cytoplasm
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
export of mrna to the cytoplasm1
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 (< 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.

ribosomal rna rrna
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
processing of rrna

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

Processing of rRNA
  • 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

slide33

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.
genetic code
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.
genetic code1

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

Genetic Code
3 possible reading frames in protein synthesis
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
trna match amino acids to codons in mrna
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
slide38
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
slide39

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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