RNA and Gene Expression BIO 224 Intro to Molecular and Cell
Biology
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RNA Molecules Three major classes Ribosomal (rRNA) make up
parts of ribosomes Messenger (mRNA) provide RNA copies of genes
Transfer (tRNA) smallest of RNAs, bring AAs to site of protein
synthesis
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Role of RNA DNA does not directly dictate protein synthesis A
molecule is needed to take information from DNA to the site of
protein synthesis RNA can be made from a DNA template SS molecule,
uses ribose as sugar, has pyrimidine U instead of T Characteristics
suggested the central dogma of the flow of genetic information in
molecular biology DNA RNA Protein mRNA molecules transcribed from
DNA, serve as template for translation of proteins
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Transcription Similar to DNA replication, but different process
Makes a complementary RNA copy of the DNA strand Entire genome
never transcribed at once Only transcribe certain genes/gene groups
at certain times RNA transcription enzymes need to recognize which
gene to transcribe and where to begin RNA polymerase enzyme
transcribes RNA from DNA template: mRNA RNA polymerase creates
nucleic acid polymer of ribonucleotides Discriminates between DNA
and RNA nucleotides Only adds ribonucleotides to polymer, creates
only RNA molecule
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4.9 Synthesis of RNA from DNA
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RNA Polymerase Recognizes start and stop points of DNA molecule
Morphologically different from DNA polymerase Both add nucleotides
to 3 OH of polymers One recognized in E coli, several identified in
eukaryotes Made of subunits, recognizes beginning of gene Binds
over region of 60 bp or so, causes DNA to locally unwind for
initiation of transcription Promoter is region upstream or at
beginning of gene that is bound Specialized short DNA sequence If
mutated, cant be bound, doesnt function
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7.1 E. coli RNA polymerase
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7.5 Structure of bacterial RNA polymerase
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Initiation Promoter sequences are upstream and downstream of
start site Near promoter is closest to transcription beginning, has
conserved sequence: TATAATA (TATA box) Far promoter has conserved
sequence: TTGACA RNA subunit allows specific binding to promoter
region sequences
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7.2 Sequences of E. Coli promoters
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Elongation After RNA polymerase binds to promoter DNA is
unwound to provide template Unwinds near beginning of gene and
provides 3OH for template 2 RNTPs are added for transcription to
begin After addition of 10 nucleotides, dissociates and allows
continuation of elongation
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7.4 Transcription by E. coli RNA polymerase (Part 1)
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7.4 Transcription by E. coli RNA polymerase (Part 2)
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Termination RNA polymerase recognizes termination signal to end
transcription Inverted repeat of GC rich area followed by poly-A
tail transcribed to form stem-loop hairpin structure Structure
disrupts RNA association with DNA template and terminates
transcription Other method involves protein Rho that binds extended
SS segments of RNA to cause termination
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7.6 Transcription termination
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Eukaryote Transcription Similar to that in prokaryotes Have
different RNA polymerases divided into classes Class I transcribes
rRNAs Class II transcribes mRNAs Class III transcribes tRNAs
Mitochondria and chloroplasts have different RNA polymerases
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Slide 18
7.11 Structure of yeast RNA polymerase II
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Eukaryote Initiation RNA polymerase recognizes promoter
sequence Upstream promoter sequence TATAA similar to bacterial TATA
box for initiation Also have downstream promoter element; some
genes use only this for initiating transcription along with Inr
Elongation occurs in similar manner to prokaryotes
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7.19 A eukaryotic promoter
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7.12 Formation of a polymerase II transcription initiation
complex (Part 1)
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7.12 Formation of a polymerase II transcription initiation
complex (Part 2)
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7.13 Model of the polymerase II transcription initiation
complex
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7.14 RNA polymerase II/Mediator complexes
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7.17 Transcription of RNA polymerase III genes
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Messenger RNA Vary in size small to very large RNA copy of
information in DNA Prokaryote mRNA is translated by ribosomes in
the cytoplasm while still being transcribed Prokaryotic mRNAs do
not exist for long Often degraded within minutes
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Eukaryote mRNA Transcribed as pre-mRNA in nucleus and processed
before export Introns removed, 5 end capped with 7-
methylguanosine, 3 end polyadenylated with poly-A tail
Polyadenylation leads to termination of transcription, important in
regulation of translation
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7.44 Processing of eukaryotic messenger RNAs
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7.45 Formation of the 3 ends of eukaryotic mRNAs
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7.47 Splicing of pre-mRNA
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7.50 Self-splicing introns
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7.52 Alternative splicing in Drosophila sex determination
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7.54 Editing of apolipoprotein B mRNA
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7.55 Regulation of transferrin receptor mRNA stability
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mRNA Translation All mRNAs translated in 5 to 3 direction
Polypeptide chains assembled from amino to carboxy terminus Each AA
specified by an mRNA codon dictated by genetic code Translation
occurs on ribosomes, needs all three types of RNAs plus
proteins
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Expression of Genetic Information DNA RNA Protein Genes
determine protein structure Proteins direct cell metabolism via
enzymatic activity Genetic information specified by arrangement of
DNA bases Proteins are polymers of 20 AAs determined by sequence
that dictates structure and function Mutation in DNA sequence leads
to AA sequence change: colinearity
Slide 37
4.8 Colinearity of genes and proteins
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The Genetic Code Genetic code allows understanding of how
sequence of 4 nucleotides is converted to 20 AAs tRNAs act as
adaptor between AAs & mRNAs during translation tRNA anticodons
pairs in complementary fashion with mRNA codons for attachment of
AA to polypeptide chain Three nucleotides specify each AA 64 codons
in genetic code: 61 code for AAs, 3 stop codons Some AAs specified
by more than one codon Nearly all organisms use same genetic
code
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Slide 40
4.11 Genetic evidence for a triplet code
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4.12 The triplet UUU encodes phenylalanine
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Transfer RNA SS, folded upon themselves into DS section with
cloverleaf structure 3 end of tRNA has CCA terminus added after
transcription, for AA binding Each AA has specific tRNA Aminoacyl
tRNA synthetase enzymes attach AAs to tRNAs in two step process
tRNAs bring individual AAs to site of protein synthesis Anticodon
loop has three base anticodon sequence specific for particular
complementary codon on mRNA tRNA anticodon base pairs with mRNA
codon to align AA Redundancy of genetic code allows less stringent
base pairing than in other processes Some AAs have more than one
codon, more than one tRNA
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4.10 Function of transfer RNA
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8.1 Structure of tRNAs
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7.43 Processing of transfer RNAs (Part 1)
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7.43 Processing of transfer RNAs (Part 2)
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8.2 Attachment of amino acids to tRNAs
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8.3 Nonstandard codon-anticodon base pairing (Part 1)
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8.3 Nonstandard codon-anticodon base pairing (Part 2)
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8.3 Nonstandard codon-anticodon base pairing (Part 3)
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8.3 Nonstandard codon-anticodon base pairing (Part 4)
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8.3 Nonstandard codon-anticodon base pairing (Part 5)
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Protein Synthesis Similar in prokaryotes and eukaryotes Occurs
on ribosomes Translation starts at specific sequence near 5 end of
mRNA, leaves 5 UTR Eukaryotic mRNAs code single polypeptide chain
(monocistronic), prokaryote mRNAs code for multiple polypeptides
(polycistronic) Both prokaryote and eukaryote mRNAs have 3
UTRs
Slide 54
8.7 Prokaryotic and eukaryotic mRNAs
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Ribosomes Sites of protein synthesis in cells Subunits
separated by ultracentrifugation, mass measured in Sphedburg units:
70S in prokaryotes, 80S in eukaryotes Made of rRNA subunits and
associated proteins Eukaryote 5S, 5.8S, 18S, 28S rRNAs all
transcribed from chromosomes Lower eukaryotes dont produce all four
Subunits transcribed as a singe unit by RNA Pol I, to precursor
that is processed into 40S and 60S subunits that make up 80S
ribosome Prokaryotes have 50S and 30S subunits of 70S ribosome
Chloroplast and mitochondrial ribosomes resemble bacterial
ribosomes Seen as a series of dots on ER, on nuclear envelope, or
in cytoplasm Cells have multiple copies of rRNA genes, actively
working cells have most ribosomes
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7.15 The ribosomal RNA gene
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7.16 Initiation of rDNA transcription
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7.42 Processing of ribosomal RNAs
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8.4 Ribosome structure (Part 1)
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8.4 Ribosome structure (Part 2)
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8.4 Ribosome structure (Part 3)
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8.5 Structure of 16S rRNA
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8.6 Structure of the 50S ribosomal subunit
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Initiation of Translation AUG is primary start codon for
translation Translation starts with AA methionine in eukaryotes and
N- formylmethionine in prokaryotes Signals for initiation codon
identification different in prokaryote and eukaryote cells
Shine-Dalgarno sequence in prokaryotes precedes mRNA initiation
sequence and base pairs with sequence near 3 terminus of 16S rRNA
Eukaryote mRNAs bound at 7-methylguanosine cap of 5 terminus before
ribosome locates initiation codon Actual initiation of translation
not entirely understood
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8.8 Signals for translation initiation
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Translation Process Divided into stages of initiation,
elongation, and termination First step of initiation is binding of
initiation factors to the small ribosomal subunit then initiator
Met tRNA and mRNA Large ribosomal subunit added to complex for
elongation to proceed Eukaryote initiation needs twelve or more
proteins, eIFs, to begin Elongation occurs after initiation, to
synthesize polypeptide chain
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8.9 Overview of translation
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8.10 Initiation of translation in bacteria
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8.11 Initiation of translation in eukaryotic cells (Part
1)
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8.11 Initiation of translation in eukaryotic cells (Part
2)
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8.11 Initiation of translation in eukaryotic cells (Part
3)
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Translation Process Three sites in ribosome for tRNA binding:
P, A, E Initiator tRNA binds to P site, second binds to A site with
help of EF and peptide bond forms Translocation moves ribosome
three nucleotides along mRNA, putting empty A site over next codon,
shifting peptidyl tRNA from A site to P site, and placing uncharged
tRNA in E site for release Elongation continues in this manner
until stop codon moves into A site Release factors recognize stop
codons and terminate translation mRNAs translated by series of
ribosomes, sometimes simultaneously
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8.12 Elongation stage of translation
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8.14 Termination of translation
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Regulation of Gene Expression All genes not expressed at all
times All genes not expressed in all cells Regulation of gene
expression is necessary to ensure production of necessary products
in correct cells at most appropriate times Gene expression is
regulated at various stages in prokaryotes and eukaryotes, with
various methods being used
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Transcriptional Regulation Mostly occurs at level of initiation
in bacteria Negative regulation: some protein products only
transcribed and translated in presence of inducers Conserves
unnecessary use of cellular energy to produce RNAs and proteins
Multiple genes expressed as operon Operator near transcription
initiation site controls transcription Repressor binds to operator
to block transcription Inducer must be present to block repressor
for transcription to occur Operator is cis-acting, repressor is
trans-acting
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Transcriptional Regulation Positive transcription control best
characterized in E. coli by effect of glucose on genes coding for
breakdown of other sugars for alternate sources of energy and
carbon If glucose available, enzymes for catabolism of other sugars
not expressed If glucose levels drop, CAP binds target sequence 60
bp upstream of transcription start site to initiate transcription
of other enzymes
Slide 79
7.7 Metabolism of lactose
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7.9 Negative control of the lac operon
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7.10 Positive control of the lac operon by glucose
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Transcriptional Regulation More complex in eukaryotes
Controlled at initiation or elongation Proteins bind regulatory
sequences and modulate RNA polymerase activity: repressors,
enhancers, promoters Modifications of chromatin structure plays a
role Cis-acting sequences regulate eukaryote expression
Slide 83
7.20 The SV40 enhancer
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7.21 Action of enhancers (Part 1)
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7.21 Action of enhancers (Part 2)
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7.22 DNA looping
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7.23 The immunoglobulin enhancer
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7.32 Action of eukaryotic repressors
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7.37 Regulation of transcription by miRNAs
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7.39 DNA methylation
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7.41 Maintenance of methylation patterns
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Translational Regulation mRNA translation regulated in
prokaryotes and eukaryotes in response to cell stress, nutrient
availability, growth factor stimulation Accomplished by repressor
proteins, noncoding microRNAs, controlled polyadenylation,
initiation factor modulation mRNA localization used in eggs,
embryos, nerve cells and moving fibroblasts, to allow protein
production in specific locations at appropriate time
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8.15 Polysomes
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8.16 Translational regulation of ferritin
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8.17 Translational repressor binding to 3 untranslated
sequences
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8.18 Localization of mRNA in Xenopus oocytes
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8.19 Regulation of translation by miRNAs
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8.20 Regulation of translation by phosphorylation of eIF2 and
eIF2B (Part 1)
Slide 99
8.20 Regulation of translation by phosphorylation of eIF2 and
eIF2B (Part 2)
Slide 100
Protein Folding and Processing Polypeptide products must be
folded into 3-D conformation to function Some protein products
consist of multiple polypeptide complexes Some proteins
additionally modified by cleavage or attachment of other functional
groups (carbohydrates and lipids)
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8.21 Action of chaperones during translation
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8.22 Action of chaperones during protein transport
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8.23 Sequential actions of chaperones
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8.27 Proteolytic processing of insulin
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8.28 Linkage of carbohydrate side chains to glycoproteins
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8.34 Palmitoylation
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Protein Regulation Regulation of enzyme activity necessary for
proper catalysis of biological reactions Accomplished through gene
expression by regulating protein production, or regulation of
protein function and activity in the cell Allosteric binding,
phosphorylation, protein degradation used to regulate enzymatic
function
Slide 108
8.36 Feedback inhibition
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8.38 Protein kinases and phosphatases
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8.39 Regulation of glycogen breakdown by protein
phosphorylation
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8.42 The ubiquitin-proteasome pathway
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8.43 Cyclin degradation during the cell cycle
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8.44 Autophagy
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