How Cells Read the Genome:
From DNA to Protein
Chapter 6
How Cells Read the Genome
► Transcription
► Translation
► Folding
of Proteins
► Evolution of the “Central Dogma”
How Cells Read the Genome
The Genomes of Multicellular Organisms: a State of Disarray!
►
►
►
►
Introns
Irregularities in Gene Density
Little organization relating to gene function
Adjacent genes often show no relatedness
How the Cells Read the Genome
The “Central Dogma”
DNA
RNA
Protein
Variations in the Central Dogma
RNA Splicing
RNA as the final gene product
Transcription
RNA vs DNA
►
►
►
►
Ribose as opposed to deoxribose sugar
Single stranded
Uracil in place of Thymine
RNA molecules w/ structural and catalytic properties
Transcription
General Features of Transcription
►
►
►
►
►
Produces RNA using one strand of DNA as template molecule
Only small portion of DNA is transcribed
Begins w/unwinding of sm portion of DNA exposing bases
Transcript elongated by complementary base pairing by RNA
polymerase
RNA transcript shorter than DNA
Transcription
RNA Polymerase
►
►
►
►
►
Transcribes DNA by catalyzing the formation phosphodiester bond btwn
nucleotides
Moves along DNA unwinding DNA just ahead of active site
Extends chain in the 5’ to 3’ direction
Hydrolysis of high-energy bonds provides energy
Many copies of RNA from same gene in small amount of time
Transcription
RNA Polymerase vs DNA Polymerse
► RNA Polymerase catalyzes addition of ribonucleotides
► Can begin RNA synthesis without primer
► Not as accurate as DNA Polymerse; RNA Polymerase 1 error/104
nucleotides vs 1 error/107 nucleotides
► Both have proof reading capability
Transcription
Different Types of RNA
► mRNA; RNA copied from genes that ultimately direct synthesis of proteins (35% total RNA)
► Final product of minority of genes is RNA itself: tRNA, rRNA, snRNA (majority
of total RNA)
► Tens of thousands of different mRNA transcripts; 10-15 copies of ea
species/cell
Transcription in Procaryotes
Start and Stop Signals embedded in DNA
►
►
►
►
►
►
►
►
Promoter= DNA sequence RNA Polymerase binds to initiate RNA synthesis
RNA Polymerase’s sigma subunit (bact) binds to promoter & opens helix
One of exposed strands serves as template
After synthsis of ~10 bases sigma subunit of RNA Polymerase disassociates
RNA Polymerase undergoes structural changes moving forward rapidly
synthesizing RNA at ~50 bp/sec
RNA Polymerase continues until termination signal of string of A-T
preceded by “hairpin loop”
Termination signal causes RNA Pol to disassociates from RNA releasing DNA
Free RNA Polym complexes once again w/ sigma subunit
Transcription in Procaryotes
Transcription
Transcription Promoter and Terminator Signals
► Heterogeneous but contain related consensus sequence recognized by
sigma subunit of RNA Pol
► Precise promoter sequence governs affinity for RNA Pol = “strength”
► Can be predicted by algorithms but need to be independently verified
► Promoters are assymetric; RNA Pol can bind in one orientation and
extend in 5’ to 3’ direction only
► Terminators more heterogeneous than promoters; ability of RNA to
fold into “hairpin loop” is most important feature
Transcription in Eucaryotes
3 Types of RNA Polymerases of similar in structure in Eucryotes
RNA Pol I- transcribes tRNA, rRNA, smRNAs
► RNA Pol II- transcribes genes encoding proteins
► RNA Pol III- transcribes tRNA, rRNA, smRNAs
►
Transcription
Transcription in Eukaryotes vs Procaryotes
►
►
Nucleosomes and higher order chromatin packaging
RNA Pol requires General Transcription Factors
Transcription
General Transcription Factors (Eukaryotes)
►
►
►
►
Assemble at promoters of all RNA Pol II transcripts
Facilitate binding of RNA Pol II
Aid in opening DNA strands for transcription to begin
Release RNA Pol from promoter into elongation mode once
transcription has begun
Transcription
5 different General Transcription Factors
Transcription
Other Proteins Required by RNA Pol in Eucaryotes
1.
2.
3.
4.
Transcriptional activators- bind to specific sequences and facilitate binding of
GTFs and RNA Pol
Mediators- enable activators to interact w/ GTFs and RNA Pol
Chromatin Remodeling Complexes- allow greater accessibility to DNA
Many proteins required to initiate transcription, > 100 subunits
Transcription
Elongation
Elongation factors= ensures that RNA Pol does not disassociate before end
of gene; assoc. w/ RNA Pol shortly after initiation of transcription
► DNA topoisomerases removes superhelical tension
► DNA gyrases uses ATP to pump supercoils into DNA
► Elongation tightly coupled to RNA processing
►
Transcription
RNA Processing
1.
2.
3.
Eucaryotic mRNA capped at 5’ end
polyadenylated at 3’ end
Introns removed
Phosphorylation of RNA tail CTD
►
►
►
consists of domain of repeated 52 times of 7 aa containing
2 serines that are phosphorylated
phosphorylation of tail promotes disassoc of RNA
Pol from proteins presents at start of transcription
allows new set of proteins that function in elongation and
pre-mRNA processeing, to assoc
Transcription
Capping of Pre-mRNAs
Capping of 5’ end w/ modified guanine nucleotide occurs after ~25 bases synthesized
3 enzymes involved in capping process
1. phosphatase removes one P’ from 5’ end of RNA
2. guanyl transferase adds GMP to 5’ end
3. methyl transferase adds methyl grp to guanosine
► Capping enzymes bind to phosphorylated tail of RNA Pol
► Cap binds CBC (cap binding complex) that facilitates RNA processing and export
►
►
Transcription
General Features of RNA Splicing
►
►
►
►
►
Involves two transesterification reactions to
join exons and remove intron in form of lariat
5 additional RNAs, > 50 proteins, and lots of
ATP required
Complexity ensures accuracy
Alternative splicing occurs in 60% of human
genes
Increase coding potential and facilitates evol
of new protein sequences
Transcription
Sequences Mark Where Splicing Occurs
Intron size varies from 10-100,000 nucleotides
► 3 conserved nucleotide sequences
5’ splice site
3’ splice site
branch points that forms base of excised lariat
►
Transcription
Spliceosome Mediates Splicing of RNA
performed primarily by 5 snRNA molec (U1,
U2, U4, U5) forming spliceosome core
► snRNA molec recognize intron-exon borders
and participate in splicing chemistry
► Spliceosome complex of RNA and protein
► More than 50 proteins invovled
►
Transcription
Mechanism of RNA Slicing
1. spliceosome recognizes splicing signals on
pre-mRNA, brings ends of intron together
2. branch point site recognized by BBP and
U2AF
3. U2 snRNP displaces BBP base pairing w/
branch point consensus seq
4. U1 snRNP base pairs w/ 5’ splice site
junction
5. U4/U6•U5 triple snRNP enters
6. RNA-RNA rearrangements disrupts U4/U6
base pairing to enable U6 to displace U1 at
5’ splice junction; U4 exits
7. U2 and U6 snRNPs in spliceosome form 3d
RNA structure bringing 5’ junction into
position near branch chain A for first
esterification
Transcription
Mechanism of RNA Slicing
8. 5’ and 3’ junctions brought together
via U5 snRNP for second esterification
9. snRNPs remain bound to lariat while
splice product released
Transcription
Spliceosome and ATP Hydrolysis
►
►
►
►
►
Not required for splicing chemistry
Needed for assembly and rearrangements
RNA helicase requiring ATP needed to break RNA-RNA interactions
All steps except assoc of BBP w/ branch chain A, and U2 w/ 5’ splice
site require ATP and other proteins
Removal of snRNP from lariat requires RNA-RNA interactions that are
dependent on ATP hydrolysis
Transcription
Assembly of Spliceosome
►
►
►
►
►
Occurs as pre-mRNA emerges from transcribing RNA Pol
Components of the spliceosome are carried on RNA Pol tail and transferred to
nascent pre-mRNA
Exon size tend to be uniform ~150 bp
Spliceosome assembly occurs co-transcriptionally, splicing sometimes occurs posttranscriptionally
Spliceosome proteins SR (rich in Ser and Arg) assemble on exon and mark off 3’
and 5’ site starting at 5’ end of mRNA, assembly occurs in conjunction w/ U1
snRNA and U2AF
Transcription
Plasticity of RNA Splicing
►
►
►
►
Splicing mech selected for flexibility
Flexibility enables cell to regulate pattern of RNA splicing
Alternative splicing when diff proteins can be made from same gene
Splicing patters regulated so diff forms of protein produced at diff
times and in diff tissues
Transcription
Self-Splicing Introns
►
►
►
►
Group I Intron= reactive G nucleotide attacks the initial phosphdiester
bound cleaved during the spicing rxn
Group II Intron= reactive A in intron seq is attaching grp, and lariat
intermediate generated
Sequence of self=splicing introns is critical; RNA folds in specific 3d
conformation that brings 5’ and 3’ junctions together and provides
precisiely positioned reactive grps to perform chemistry
Pre-mRNA splicing mech evolved from Grp II Splicing
spliceosomal snRNPs took over structural and chemical roles of Grp II
Introns so sequence constraints no longer needed
Transcription
Group I Introns
Group II Introns
Transcription
Processing of 3’ End of pre-mRNA
Termination signs are transcribed into RNA and recognized proteins as RNA
Pol transcribes thru them
► CstF (cleavage stimulating factor F) and CPSF (cleavage and processing
specificity factor) proteins assoc w/ RNA Pol tail transferred to RNA as it
emerges
►
Transcription
Processing the 3’ End of the Pre-mRNA
►
Additional proteins assemble w/ CstF and CPSF to
perform processing:
1. RNA is cleaved
2. Poly-A-Polymerase adds ~200 A’s to 3’ end of
cleaved product
3. RNA Pol II continues to transcribe after premRNA has been
cleaved; several 100 bases before falls off
template and transcription terminates
4. RNA downstream of cleavage is degraded
Transcription
Selective Export of Mature mRNAs from Nucleus
How does cell distinguish btwn rate mature mRNA and debris from mRNA
processing?
► Export highly selected and coupled to correct mRNA processing
► mRNA exported only if appr set of proteins are bound including: 1) cap binding complex
2) snRNP proteins absent 3) proteins that mark complete splicing
Selective Export of Mature mRNAs from Nucleus
Transcription
hnRNPs= heterogeneous nuclear ribonuclear proteins, most abundant
proteins that assemble on pre-mRNA as it emerges from RNA Pol
► Some hnRNPs remove hairpin helices from RNA so that splicing and other
signals on RNA can be read
► hnRNPs excluded from exons, remain on excised introns marking them
for nuclear retention and/or destruction
► Some reamin bound to fully processed mRNA and accompany them to
cytoplasm
Transcription
Most of the RNA in the Cell Performs a Catalytic or Structural Function
► ~80% of total RNA is rRNA
► 3-5% of total RNA mRNA
► rRNA transcribed by RNA Pol I (which has no C-terminal tail)
► rRNA is neither capped or polyadenylated
► RNA components of ribosome are final gene products; growing cell syn
~10 million of ea type of rRNA ea cell generation
Transcription
rRNA Genes
►
►
►
Mammalian cells contain 10 million ribosomes
Multi-copy genes
E. coli has 7 copies of its rRNA
Humans ~200 copies on 5 chromosomes
Xenopus ~600 copies single cluster on 1 chromosome
4 types of eucaryotic rRNAs ea present in one copy/ribosome
18S, 5.8S and 28S encoded by single lg precursor RNA-chemically modified
5S rRNA syn from separate cluster by Pol III- not chemically modified
Transcription
Chemical Modification of Lg rRNA Precursor
► 100 methylations of 2’-OH
► 100 isomerizations of uridines
► Function of chem. modification unknwn but may
facilitate folding or assembly
► snoRNAs= small nucleolar RNAs guide in chemical
modification and cleavage of precursor rRNA
► snoRNAs encoded in introns, esp those of ribosomal
proteins, function in nucleolus
Transcription
Nucleolus as a Ribosome Producing Factory
site for processing rRNAs and assembly into
ribosomes
► lg aggregate of macromolecules including: rRNA
genes, precursor rRNAs, mature rRNAs, rRNA
processing enzymes, snoRNPs, ribosomal protein
subunits, and some partially assembled ribosomes
► Size varies and reflects number of ribosomes cell is
making; may occupy 25% of nuclear vol
► Also site where other RNAs produced and other
RNA-protein complexes assembled (tRNAs,
snoRNAs, U6 snRNP, telomerase)
►
Translation
From RNA to Protein
►
►
►
►
Genetic code dictates how mRNA translated into aa sequence of protein
Nucleotides read consecutively in grps of 3 (condons)
Degenerate genetic code; 64 codons specify 20 aa
6 possible reading frames
Translation
tRNA as an Adaptor Molecule
Recognizes and binds to codon and appr. aa
► ~80 nucleotdies
► Folds into clover leaf and then into L-shaped structure
► Two regions of unpaired nucelotdies at ends of L shaped molecule essential to function
1. anticodon= set of 3 consecutive nucleotides pairs w/complementary codon
on mRNA
2. short ss region near 3’end where aa attaches
►
Translation
Redundancy/Degeneracy of Genetic Code
More than one tRNA for many of the aa
► Some tRNAs base pair w/ more than one aa; Wobble Position
►
Translation
tRNAs
►
►
►
►
►
►
Number and kinds of tRNAs varies across species ie:
humans have 497 tRNA genes w/ only 48 diff
anticodons represented
Transcribed by RNA Pol III
Some transcripts are spliced via cut-and-paste
mechanism catalyzed by proteins (no lariat) occurs
when tRNA is properly folded in cloverleaf conf
Extensive modifications > 50 different kinds
1 modification/10 nucleotides
Modifications essential to:
1) accuracy of tRNA attaching to correct aa
2) recognition of mRNA codon by tRNA anticodon
Translation
Aminoacyl-tRNA Synthetases
Couple aa to appr set of tRNAs
► Aa specific
► Enzymatic reaction attaches aa to 3’ end of tRNA requires ATP
► High energy bond produced btwn aa and tRNA later used to covalently link aa to
growing polypeptide chain
►
Translation
Editing by tRNA synthetases
►Selects correct aa in two step process:
1. correct aa highest affinity for active site
2. aa forced into second pocket whose
dimensions exclude correct aa
►Those that enter second editing site
are hydrolyzed from AMP- hydrolytic editing
►Raises overall accuracy of tRNA charging to
1 mistake/40,000 couplings
►Synthetase must also recognize correct tRNAs
Translation
Ribosome Structure: rRNA molecules + 50 different proteins
Translation
Ribosome Structure and Function
Large and Small Subunits; rRNA sequence highly conserved
► 66% RNA; 33% protein
► rRNA responsible for:
structure
positioning tRNAs on mRNA
catalytic activity in forming peptide bond
►
Translation
mRNA Decoded on Ribosomes
►
►
►
►
►
Ribosomal subunits assemble on mRNA near 5’ end
Ribosome translates mRNA in aa sequence using tRNAs as adaptors to add
aa in correct sequence to end of growing polypeptide chain
aa linked by peptide bond formation
2 aa added/sec eucaryotic cell; 20 aa/sec added by bact
4 impt ribosomal binding sites
Translation
Major Steps in Translation
1.
Ribosomal Assembly and Initaition
2.
Elongation
3.
Termination and Release of Nascent Polypeptide
Translation
Ribosomal Assembly
1.
2.
3.
4.
5.
Initiation tRNA-Met & eIFs bind sm rRNA subunit
Sm rRNA subunit binds to 5’ end of mRNA
recognizing CAP and 2 eIFs (eucaryotes)
Sm rRNA scans for AUG start
eIFs dissociate and lg rRNA binds
Initiator tRNA-met now in P-site leaving A-site
vacant for incoming aminoacyl rRNA to start
protein synthesis
Translation
Procaryotes have Shine Delgarno Sequence
Specific sequence few nucleotides upstream of AUG start
► AGGAGGU
► Binding site positions sm rRNA subunit at AUG start
► Bacterial mRNAs can be poly-cistronic; eucaryotes monocistronic
►
Translation
Elongation
1.
2.
3.
tRNA carrying next aa in chain binds
to ribo A-site base pairing w/ mRNA
codon
Peptide Bond Formation= carboxyl end
released from tRNA at P-site and
joined to free amino grp of aa linked
to tRNA at A site forming new peptide
bond
Translocation= conformational chgs
move mRNA 3 nucleotides along ribo
resetting ribo for next acyl tRNA
Translation
Two Impt Elongation Factors Drive Translation
►
►
EF-Tu Mechanism- resp. 99% accuracy
a. Chged tRNA enters ribo bound to GTP form
b. Codon recognition triggers GTP hydrolysis
c. EF-Tu dissociates from ribo w/out tRNA
d. Introduces 2 short delays btwn codonanticodon pairing and chain elongation
EF-G binds in or near A site hydrolyzes GTP whose energy
accelerates movement of bound tRNAs in A/P and P/E
hybrid states
Translation
Termination
►
►
►
►
►
Stop codons: UAA, UAG, UGA
Stop codons recognized by “release factor” proteins
Release factors cause peptidyl transferase to catalyze
addition of water to peptidyl tRNA
Hydrolysis frees COOH end of polypeptide chain from
attachment to tRNA
Ribosomal dissassembly
Translation
Proteins are Made on Polyribosomes
Syn of avg protein varies 20 sec to 2-3 min
► mRNAs translated in form of polyribosomes w/ ribos spaced > 80
nucleotides
► Transcription and translation occur simultaneously in procaryotes
►
Translation
Quality Control of Translation
►
►
►
►
Accuracy ~1 mistake/104 aa
Speed of translation 20 aa incorporated/sec in pro. vs 2 aa/sec euk.
Protein synthesis consumes more energy than any other biosynthetic
process; 4 ATP equivalents/ aa
Quality control mech to ensure mRNA is complete recognition of Cap
and poly A tail by initiation complex
Translation
Antibiotics
►Inhibitors of bacterial protein synthesis are effective antibiotics
►Specificity of antibiotics useful for molecular cell biology studies
Life and Death of Proteins
Protein Maturation
Protein folding begins while protein is being synthesized
► Maturation:
unique 3d structure
bind sm molecules
modifications
assemble w/ other proteins
►
Life and Death of Proteins
Protein Folding
► Steps in protein folding:
1. molten globule
2. slow phase
► Most of folding complete by time
released from ribosome
► Molecular chaperones held guide
folding of many proteins
Life and Death of Proteins
Molecular Chaperonins
►
►
►
►
►
Two major families: hsp60 & hsp70
Affinity for exposed hydrophobic
patches
Massage protein into folded
conformation via ATP hydrolysis
Hsp70 acts early in life of protein;
binds to stretch of 7 hydrophobic aa
before protein leaves ribo
Hsp60 acts later in proteins life, forms
barrel into which proteins fed
Life and Death of Proteins
Proteosome
► Protein disposal apparatus dispersed throughout cytosolo
► Hollow cylinder of proteases form stack of 4 heptameric rings
► Ends composed of protein complex of ~20 polypeptides, 6 of which
hydrolyze ATP to unfold proteins and move them into proteosome
► Acts on proteins marked by ubiquitin
Life and Death of Proteins
Ubiquitin conjugating system
Multiubiquitin chain on target proteins recognized by receptors on proteosome
► Targeted proteins: misfolded, oxidized, or other abnormal aa
► Linear series of ubiquitin conjugates linked via lysine on target protein
►
Life and Death of Proteins
Regulated Destruction
►
►
►
Activation of compments in proteolytic pathway E1, E2, E3
Degradation signals created in response to intracellular or extracellular signals
N-terminal rule
12 destabilizing aa including Arg, Lys, His, Leu, Asp Try, Tyr, Asp, Glu, Asn, Gln