From DNA to RNA The RNA world MB 207 – Molecular Cell Biology.
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Transcript of From DNA to RNA The RNA world MB 207 – Molecular Cell Biology.
From DNA to RNAThe RNA world
MB 207 – Molecular Cell Biology
TheNucleolus
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?
• 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
• 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
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
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
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
DNA Transcription
• 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 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)
Transcription – InitiationConsensus sequences found in the vicinity of eukaryotic RNA Polymerase II start points
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
Initiation of transcription of a eukaryotic gene by RNA polymerase II
A. The promoter contains a DNA sequence called the TATA box
B. TATA box is recognized and bound by transcription factor TFIID, which enables the binding of TFIIB.
C. DNA distortion produced by the binding of TFIID is not shown.
D. The rest of the general transcription factors, RNA polymerase, assemble at the promotor.
E. 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 cellBinds to short sequences in DNA, acting from a distance (thousands of nt pairs)
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 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
Transcription - Elongation
Steps of elongation:
1. Unwinding of DNA in front of the enzyme
2. Synthesis of RNA
3. RNA proofreading (one mismatch consists thousand nucleotides)
a. pyrophosphorolytic editing
b. hydrolytic editing
1. Dissociation of RNA
2. Re-annealing of DNA behind the enzyme
mRNA 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 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
Capping is carried out by 3 enzymes:
1. Phosphatase
2. Guanyl transferase
3. Methyl transferase
The structure of the cap at 5’ end
The reaction that cap the 5’ end of each RNA molecule synthesized by RNA polymerase II
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
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)
• 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.
1. A specific Adenine nucleotide in the intron sequence attacks the 5’ splice site
2. Cut the sugar phosphate backbone
3. 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.Creating a loop in the
RNA molecule
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/UR=A/G
For example: RNA splicing
RNA splicing is performed by the spliceosome
• RNA splicing:– Spliceosome is 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• 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
Transcription - termination
Terminator: trigger the elongation polymerase to dissociate from the DNA and release the RNA chain.
Types of bacterial terminators:
a. Rho-indipendent terminators (intrinsic terminators – without involvement of other factors)
b. 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
• 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 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.
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 3’end of the growing RNA chain
Transcription cycleInitiation•Formation of a closed complex•Transition to an open complex•Promoter escape
Elongation•Unwinding of DNA in front of the enzyme•Synthesis of RNA•RNA proofreading•Dissociation of RNA•Re-annealing of DNA behind the enzymeTermination•Rho-independent terminators•Rho-dependent terminators
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• 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
The chemical modification and nucleolytic processing of an eukaryotic 45S precursor rRNA molecule into 3 separate ribosomal RNAs
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
– 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
• 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 mitochondriao 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 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 proteinStart codon bacteria
Stard codon eukaryotic
5’-AUG-3’ 5’-AUG-3’
5’-GUG-3’
5’-UUG-3’
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
• 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
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
• 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
tRNA
– 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