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Transcription…. Transcription – the process of copying a DNA template into an RNA strand Accomplished via DNA dependent RNA polymerase (aka RNA polymerase) Similar to DNA replication but: uses RNA polymerase RNA product is displaced nearly immediately from template is less accurate

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Transcription

Transcription…

  • Transcription – the process of copying a DNA template into an RNA strand

  • Accomplished via DNA dependent RNA polymerase (aka RNA polymerase)

    • Similar to DNA replication but:

    • uses RNA polymerase

    • RNA product is displaced nearly immediately from template

    • is less accurate

    • is not interested in the whole genome, only sections


Transcription1

Transcription…

  • By the end of this series, you should be able to explain much of this animation

  • http://www.as.wvu.edu/~dray/219files/Transcription_588x392.swf


Transcription

  • Bacterial RNA polymerase 50 - 100 nucleotides/sec

  • Most genes are transcribed simultaneously by numerous polymerases

  • Polymerase moves along DNA in 3' —> 5' direction

  • Complementary RNA constructed in ____ direction

    • RNAn + NPPP —> RNAn+1 + PPi


Transcription2

Transcription

  • How does the polymerase know where to start?

    • Promoter = the assembly point for the transcription complex

  • RNA polymerases cannot recognize promoters on their own - transcription factors

    • Transcription factors - enzymes have evolved to recognize (physically interact with) specific DNA sequences and with other proteins


Transcription

Prokaryotic Gene Regulation

  • Preliminaries

  • DNA binding proteins

  • Minor vs. Major grooves

  • Bases are exposed in the grooves, providing binding sites

  • Each bp provides characteristic binding sites

A – H acceptor, D – H donor, H – nonpolar H, M – methyl group


Transcription

Prokaryotic Gene Regulation

  • Protein-nucleic acid binding

  • Most proteins to be discussed bind specific DNA sequences

  • Most commonly via α-helix insertion into major groove(s)

    • H donor/acceptors

    • + charge on helix interaction with phosphate backbone

  • Helix-turn-helix motif

    • 1st DNA binding domain identified


Transcription

Transcription

  • Three-phase progression

  • Initiation –

    • Promoter recognition and binding

    • DNA melting

    • Transcript initiation

  • Elongation

    • mRNA produced

  • Termination

    • Polymerase and RNA released


Transcription

Transcription

  • Bacterial transcription

  • Promoters

  • Core enzyme (α2ββ’ω) will begin transcription just about anywhere

  • σ provides specificity

  • Core + σ = holoenzyme

  • E. coli  σ70

  • σ70 promoters

    • 2 conserved sequences of 6 bp each

    • -10, -35


Transcription

Transcription

  • Bacterial transcription

  • Promoters

  • Not all -10 and -35 sequences are identical

  • Consensus sequences

    • -35 – TTGACA

    • -10 - TATAAT


Transcription3

Transcription

  • Prokaryotic Transcription

  • Bacterial promoters are located just upstream of the RNA synthesis initiation site

    • The nucleotide at which transcription is initiated is called +1; the preceding nucleotide is –1

    • DNA preceding initiation site (toward template 3' end) are said to be upstream

    • DNA succeeding initiation site (toward template 5' end) are said to be downstream


Transcription4

Transcription

  • Prokaryotic Transcription

  • One RNA polymerase with 5 subunits tightly associated to form core enzyme

  • Core enzyme minus sigma (σ) factor will bind to any DNA.

    • By adding σ, RNA pol will bind specifically to promoters (-10 & -35 sequences)

  • Transcription begins de novo

  • Requires stable interaction b/t base and template while second base is recruited


Transcription

Transcription

  • Bacterial transcription

  • Three models of initial transcription

  • How to explain?

  • Transient excursion – Pol moves along DNA chain

  • Inchworming –

  • Scrunching – DNA pulled in

  • Experiments demonstrate:

    • Pol remains stationary on promoter

    • Pol subunits remain stationary relative to one another

  • Suggests that ‘scrunching’ is it

    • As DNA/RNA ‘piles up’ inside the polymerase, it creates pressure which contributes to forcing the polymerase off of the promoter


Transcription

Transcription

  • Bacterial transcription

  • Several false starts before elongation begins

    • ≤ 9bp

  • ‘Escape’ - transcription of 10+ bp leading to elongation phase

  • 10+ bp too long to remain hybridized to tempate

  • Pol must break interactions with promoter and regulatory factors

  • σ ¾ linker must be ejected from RNA exit channel

  • ‘Scrunched’ DNA likely provides the energy to break pol-promoter interactions and dislodge σ


Transcription

Transcription

  • Bacterial transcription

  • Termination

  • Rho (ρ) dependent

  • ATPase activity leads to translocation along transcript


Transcription

Transcription

  • Bacterial transcription

  • Termination

  • ρ-dependent

  • Rut (ρ utilization) sites – not well-defined but sequence dependent

  • http://archive.microbelibrary.org/microbelibrary/files/ccImages/Articleimages/sandrin/rhodependent.htm

  • http://highered.mcgraw-hill.com/sites/dl/free/0072835125/126997/animation21.html


Transcription

Transcription

  • Bacterial transcription

  • Termination

  • ρ-independent

  • Two sequence elements

    • Inverted repeat

    • ~8 A:T stretch

  • Hairpin formation disrupts polymerase function

  • A:U binding weak


Transcription5

Transcription

  • Eukaryotic vs. Prokaryotic Transcription

  • Much of what we know is derived from studies of RNA pol II from yeast

    • 1. Seven more subunits than its bacterial RNA pol

    • 2. The core structure & the basic mechanism of transcription are virtually identical

    • 3. Additional subunits of eukaryotic polymerases are thought to play roles in the interaction with other proteins

    • 4. Eukaryotes require a large variety of accessory proteins or transcription factors (TFs)


Transcription

Transcription

  • Eukaryotic transcription

  • Contrasts with prokaryotes

    • Three polymerases

      • RNA polymerase I (RNA pol I) - synthesizes the larger rRNAs (28S, 18S, 5.8S)

      • RNA polymerase II (RNA pol II)- synthesizes mRNAs & most small nuclear RNAs (snRNAs & snoRNAs)

      • RNA polymerase III (RNA pol III) - synthesizes various small RNAs (tRNAs, 5S rRNA & U6 snRNA)

    • Multiple general transcription factors (GTFs)

    • Additional regulatory elements (enhancers, chromatin modifiers, silencers, insulators, etc.)

  • RNA pol II – transcribes mRNA


Transcription

Transcription

  • Eukaryotic transcription

  • RNA pol II core promoters

  • Four different sequence elements

    • TFIIB recognition element (BRE box)

    • TATA box

    • Initiator box (Inr)

    • Downstream promoter elements (DCE, DPE, MTE)

  • Various combinations in most Pol II promoters

    • All not necessary

    • Inr most common


Transcription

Transcription

  • Eukaryotic transcription

  • Preinitiation complex formation

  • TBP component of TFIID

  • β sheet inserted into minor groove, bending DNA 80°

  • Specificity via phenylalanine chain intercalation on flanks of sequence


Transcription

Transcription

  • Eukaryotic transcription

  • Preinitiation complex formation

  • TFIID TFIIA TFIIB TFIIF TFIIE TFIIH


Transcription

Transcription

  • Eukaryotic transcription

  • Preinitiation complex formation

  • TFIID – initial binding and recruitment

  • TFIIA – clamp

  • TFIIB - recruitment of pol II, may insert into RNA exit channel

  • TFIIF – stabilize complex, required for recruitment of TFIIE/H

  • TFIIE – recruit and regulate TFIIH

  • TFIIH – kinase

    • Largest GTF

    • Phosphorylates carboxy-terminal domain (CTD) of pol II

    • http://www.crocoduck.bch.msstate.edu/BCH4713/Transcription.wmv


Rna processing mrna

RNA processing – mRNA

  • 5’ cap

    • The raw transcript will be immediately degraded in the cytoplasm so it must be marked and protected

  • Possible/known functions of 5’ cap

    • May prevent exonuclease digestion of mRNA 5' end,

    • Aids in transport of mRNA out of nucleus

    • Important role in initiation of mRNA translation


Rna processing mrna1

RNA processing – mRNA

  • mRNA processing – Splicing

  • Requires break at 5' & 3' intron ends (splice sites) & covalent joining of adjacent exons (ligation)

    • http://www.as.wvu.edu/~dray/219files/mRNASplicingAdvanced.wmv

  • Why introns?

    • Disadvantages – extra DNA, extra energy needed for processing, extra energy needed for replication

    • Advantages – modular design allows for greater variation and relatively easy introduction of that variation


  • Rna processing mrna2

    RNA processing – mRNA

    • mRNA processing – Splicing

    • Splicing MUST be absolutely precise

    • Most common conserved sequence at eukaryotic exon-intron borders in mammalian pre-mRNA is G/GU at 5' intron end (5' splice site) & AG/G at 3' end (3' splice site)


    Rna processing mrna3

    RNA processing – mRNA

    • mRNA processing – Splicing

    • Sequences adjacent to introns contain preferred nucleotides that play an important role in splice site recognition


    Rna processing mrna4

    RNA processing – mRNA

    • mRNA processing – Splicing

    • Nuclear pre-mRNA (common)

      • snRNAs + associated proteins = snRNPs

        • snRNAs – 100-300 bp

        • U1, U2, U4, U5, U6

      • 3 functions for snRNPs

        • Recognize sites (splice site and branch point site)

        • Bring these sites together

        • Catalyze cleavage reactions

      • Splicosome – the set of 5 snRNPs and other associated proteins

      • Summary movie available at:

      • http://www.as.wvu.edu/~dray/219files/mRNAsplicing.swf


    Rna processing mrna5

    RNA processing – mRNA

    • mRNA processing – Splicing

    • 1. U1 and U2 snRNPs bind via complementary RNA sequences

    • Note the A bulge produced by U2

    • U2 is recruited by proteins associated with an exon splice enhancer (ESE) within the exon


    Rna processing mrna6

    RNA processing – mRNA

    • mRNA processing – Splicing

    • 2. U2 recruits U4/U5/U6 trimer

    • U6 replaces U1, U1 and U4 released

    • U5 binds to upstream exon


    Rna processing mrna7

    RNA processing – mRNA

    • mRNA processing – Splicing

    • 3. U6 catalyzes two important reactions

      • Cleavage of upstream exon from intron (bound to U5)

      • Lariat formation with A bulge on intron

    • Exons are ligated

    • U2/U5/U6 remain with intron


    Transcription

    DNA/RNA Structure

    OH

    OH

    NB

    NB

    NB

    NB

    DNA

    RNA

    OH

    O

    OH

    OH

    P

    O

    C

    OH

    O

    P

    P

    P

    O

    O

    O

    C

    C

    C

    O

    OH

    OH


    Rna processing mrna8

    RNA processing – mRNA

    • mRNA processing – Splicing

    • http://www.crocoduck.bch.msstate.edu/BCH4713/ch13_group_II_introns.html

    • Several lines of evidence suggest that it is the RNA in the snRNP that actually catalyzes the splicing reactions

      • 1. Pre-mRNAs are spliced by the same pair of chemical reactions that occur as group II (self-splicing) introns

      • 2. The snRNAs needed for splicing pre-mRNAs closely resemble parts of the group II introns

    • Proteins likely serve supplemental functions

      • 1. Maintaining the proper 3D structure of the snRNA

      • 2. Driving changes in snRNA conformation

      • 3. Transporting spliced mRNAs to the nuclear envelope

      • 4. Selecting the splice sites to be used during the processing of a particular pre-mRNA


    Transcription

    Transcription

    • Polyadenlyation

    • The poly(A) tail – 3' end of most mRNAs contain a string of adenosine residues (100-250) that forms a tail

      • Protects the mRNA from degradation

      • AAUAAA signal ~20 nt upstream from poly(A) addition site

      • Poly(A) polymerase, poly(A) binding proteins, and cleavage factors are involved

      • Simple animation on website


    Transcription

    Transcription

    • Eukaryotic transcription

    • Termination

    • Torpedo model

    • Post-cleavage RNA is uncapped

    • Recognized by Rnase (Xrn2 in humans)

    • Again, CTD bound

    • Highly processive, displaces Pol II


    Transcription

    Transcription

    • Eukaryotic RNA pol I

    • rRNA transcription

    • SL1 = TBP + three TAFs


    Transcription

    Transcription

    • Eukaryotic RNA pol III

    • tRNA and 5S transcription

    • Internal promoter sequences


    Transcription

    Prokaryotic Gene Regulation

    • Principles

    • Activators and repressors

    • DNA binding proteins

    • Primary level of action – transcription initiation

    • Activators enhance RNA polymerase binding

    • Repressors block RNA polymerase binding

    • Many regulatory proteins work via allostery


    Transcription

    Prokaryotic Gene Regulation

    • Principles

    • Action at a distance

    • Interaction b/t distantly binding proteins accommodated by DNA loops

    • Often aided by architectural proteins


    Transcription

    Prokaryotic Gene Regulation

    • Control of Gene Expression: Prokaryotes

      • The operon - in bacteria, genes for enzymes of metabolic pathway are usually clustered in functional complex under coordinate control

      • Terminology:

      • 1. Genes - code for operon enzymes; usually adjacent to each other; turn on one, turn on all

      • 2. Promoter

      • 3. Operator – typically resides adjacent to or overlapping with the promoter; repressor protein binding site

      • 4. Repressor - gene regulatory protein; binds with high affinity to operator

      • 5. Regulatory gene - encodes repressor protein


    Transcription

    Prokaryotic Gene Regulation

    • The operon…

      • The repressor is key to operon expression; if it binds to operator; it shields promoter from polymerase & prevents transcription

        • 1. Repressor binding to operator depends on conformation, which is regulated by a key compound in the metabolic pathway (lactose or tryptophan)

        • 2. Concentration of key metabolite determines if operon is active or inactive at any given time


    Transcription

    Prokaryotic Gene Regulation

    • The lac operon…

      • An inducible operon – the presence of a key substance induces the transcription of the genes.

      • Regulates production of the enzymes needed to degrade lactose in bacterial cells

        • Genes in the lac operon

        • 1. z gene - encodes β-galactosidase

        • 2. y gene - encodes galactoside permease; promotes lactose entry into cell

        • 3. a gene - encodes thiogalactoside acetyltransferase; its physiological role is unclear


    Transcription

    Prokaryotic Gene Regulation

    • Prokaryotic gene expression…

      • Lactose (disaccharide) - made of glucose & galactose

      • Oxidation provides the cell with metabolic intermediates & energy

      • The β-galactoside linkage is broken in the first step of catabolism - β-galactosidase


    Transcription

    Prokaryotic Gene Regulation

    • Control of Gene Expression: Prokaryotes

      • Prokaryotes live in constantly changing environment

      • It is advantageous for cells to use available resources in most efficient way so regulate responses

      • Thus, they respond by selective gene expression

      • If lactose is absent—> β-galactosidase not needed & not present (<5 copies of enzyme, 1 of the corresponding mRNA)

      • If lactose is present —> enzyme levels rise ~1000-fold in a few minutes; lactose has induced the synthesis of β-galactosidase


    Transcription

    Prokaryotic Gene Regulation

    • The lac operon…

      • 1. If lactose is present in medium, it enters cell, binds lac repressor, changing its shape. Lactose acts as an inducer

      • 2. Lactose-bound repressor cannot bind operator DNA

      • 3. If lactose levels fall, it dissociates from repressor, changing repressor back to active shape

      • 4. Repressor binds operator and physically blocks polymerase from reaching structural genes, turns off transcription

      • Lac operon movie


    Transcription

    Prokaryotic Gene Regulation

    • The lacoperon

    • Lac repressor binding


    Transcription

    Prokaryotic Gene Regulation

    • The lacoperon

    • Both bind using similar amino acid motifs

    • Helix-turn-helix

    • Recognition helix interacts with DNA

    • λ-repressor example


    Transcription

    Transcription...

    • Control of Gene Expression: Prokaryotes

      • Tryptophan - essential amino acid needed for protein synthesis; if it is not in the growth medium, it must be produced by bacterium

        • 1. In its absence, cells contain enzymes & their mRNAs needed to make tryptophan

        • 2. If tryptophan is available in medium, bacteria don't need enzymes to make it; the genes of those enzymes are repressed within a few minutes & the production of the enzymes stops


    Transcription

    Transcription...

    • The trp operon…

      • A repressible operon – the presence of a key substance represses the transcription of genes.

      • Repressor is active only if bound to specific factor which functions as a co-repressor (like tryptophan)


    Transcription

    Transcription...

    • The trp operon…

      • 1. Without tryptophan, operator site is open to binding by RNA polymerase

      • 2. Production of enzymes that synthesize tryptophan

      • 3. When tryptophan is available, enzymes of tryptophan synthetic pathway are no longer needed

      • 4. Increased tryptophan concentration leads to formation of tryptophan-repressor (active repressor)

      • 5. Repressor binds DNA at operator, blocks transcription

      • http://bcs.whfreeman.com/thelifewire/content/chp13/1302002.html


    Transcription

    The Cell Nucleus…

    • Control of Gene Expression: Eukaryotes

      • A single human cell contains enough DNA (6 billion bp) to encode several million different polypeptides

        • 1. Most of this DNA does not actually code for proteins, mammalian genomes are thought to contain ~30,000 protein-coding genes

        • 2. A typical mammalian cell may make ~5,000 different polypeptides at any given time

        • 3. Many of these are made by virtually all cells of the organism

        • 4. Cells also make proteins unique to its differentiated state; giving the cell its unique characteristics

        • 5. Regulating eukaryotic gene expression is an extremely complex process, just starting to be understood


    Transcription

    The Cell Nucleus…

    • Control of Gene Expression: Eukaryotes

      • Three levels of control

        • Trascriptional

        • Processing

        • Translational


    Transcription

    The Cell Nucleus…

    • Regulatory Regions

      • The regulatory region of a gene can be thought of as an integration center for that gene's expression

        • The extent to which a given gene is transcribed depends upon particular combination of TFs bound to its upstream regulatory elements

          • 1. Roughly 5 – 10% of genes encode TFs

          • 2. Thus, a nearly unlimited number of possible combinations of interactions among TFs is possible

          • 3. Complexity of interactions is revealed in marked variation in gene expression patterns between cells of different type, different tissue, different developmental stage & different physiological state


    Transcription

    The Cell Nucleus…

    • Regulatory Regions

      • Promoter elements – regions upstream of a gene that regulate the initiation of transcription.

      • Most eukaryotic promoter elements can be roughly divided in to ‘proximal’ and ‘distal’

      • Proximal promoter elements (-50 to -200bp):

        • TATA box –

          • Consensus sequence – TATAAA

          • Usually at ~-30

        • CAAT box –

          • Consensus sequence – CAAT

          • Usually ~-70

        • GC box –

          • Consensus sequence – GGGCGG

          • Often multiple copies within 100 bp upstream of start codon


    Transcription

    The Cell Nucleus…

    • Regulatory Regions

      • Proximal promoter elements (-50 to -200bp):

        • TATA box –

          • Site of assembly of the transcription complex:

          • RNA polymerase II, all necessary transcription factors

        • CAAT box and GC box –

          • Regulate the frequency of transcription via binding of transcription factors


    Transcription

    The Cell Nucleus…

    • More Regulatory Elements

      • Enhancers

      • Raise transcription rates above the basal level

        • 1. Have a unique property: they can be moved experimentally from one place to another within a DNA molecule (even be inverted) without affecting the ability to stimulate transcription

        • 2. Deletion of an enhancer can decrease the level of transcription by 100-fold or more

        • 3. Some enhancers are located thousands or even tens of thousands of base pairs upstream or downstream from the gene whose transcription they stimulate


    Transcription

    The Cell Nucleus…

    • More Regulatory Elements

      • Enhancers

      • Thought to stimulate transcription by influencing events that occur at core promoter

        • A. Enhancers & core promoters can be brought together via DNA loops

        • Remember movie from chapter 11?

        • B. What prevents enhancer from binding to inappropriate promoter located even farther downstream?

          • 1. insulators

          • 2. insulator sequences may bind to proteins of nuclear matrix; DNA segments between insulators correspond to looped domains of chromatin

      • How do transcriptional activators bound at enhancer stimulate transcription initiation at core promoter?

        • coactivators; 2 basic types:

          • 1. Those that interact with components of the basal transcription machinery (general TFs & RNA polymerase II) - lead to assembly of preinitiation complex & initiation of RNA synthesis

          • 2. Those that act on chromatin, converting it from a state relatively inaccessible to transcription machinery to a much more transcription-friendly state


    Transcription

    The Cell Nucleus…

    • Control of Gene Expression: Eukaryotes

      • Transcriptional control is orchestrated by actions of a large number of proteins called transcription factors (TFs);

      • 1. General TFs - bind at core promoter sites in association with RNA polymerase

      • 2. Sequence-specific TFs - bind to various regulatory sites of particular genes; they either stimulate (transcriptional activators) or inhibit (transcriptional repressors) transcription of adjacent genes


    Transcription

    Eukaryotic Gene Regulation

    • DNA binding domains

    • Dimerization common

    • Bacterial and eukaryotic DNA binding domains can often be interchanged

    • Homeodomains

      • Helix-turn-helix

      • All eukaryotes


    Transcription

    Eukaryotic Gene Regulation

    • Zinc binding domains

    • Zinc finger/zinc cluster

      • Cys/His

      • TFIIIA

      • Glucocorticoid receptor

    TFIIIA


    Transcription

    Eukaryotic Gene Regulation

    • Helix-loop-helix

    • Long helix for DNA binding

    • Shorter helix for dimerization or other function


    Transcription

    Eukaryotic Gene Regulation

    • HMG box

    • Three helices in boomerang shape

    • DNA bending

    • Architectural factors

    • SRY gene and testosterone and XY females

    • HMG factor binds upstream of SRY to increase testosterone production

    • Activation domains

    • Not well-defined

    • Often grouped by amino acid content


    Transcription

    Eukaryotic Gene Regulation

    • Signal transduction

    • any process by which a cell converts one kind of signal or stimulus into another

    • Often in response to external stimuli

    • Many, many different pathways specific to particular situations

    • Some general commonalities:

      • Ligand usually initiates the pathway

      • Cell surface receptors

      • Path is often a cascade of kinases

      • Sometimes, activating region is “unmasked”

      • Sequestering inactive activators and repressors in the cytoplasm

      • Sometimes, participants come in pieces that are later combined

    • Examples follow


    Transcription

    Eukaryotic Gene Regulation

    • Signal transduction

    • Example – STAT (signal transducer and activator of transcription)

    • Cytokine – intercellular signaling molecule

    • SH2 domain of STAT is variable and different STATs will target different genes

    • Three examples on website


    Transcription

    Eukaryotic Gene Regulation

    • Signal transduction

    • Example – Ras/MAPK (mitogen activated protein kinase)

    • GDP-GTP exchange induces conformational change in Ras

    • Kinase cascade with MAPK proteins

    • Example animations on website (with cool sound effects)


    Transcription

    Eukaryotic Gene Regulation

    • Signal transduction

    • Example – A generalized cAMP pathway

      • G protein

        • Inactive – trimer bound to GDP

        • Activation - GDP replaced by GTP

        • Active – monomer + GTP, dimer

        • Largest superfamily of human proteins (1000+)

      • cAMP – a common second messenger generated by adenylatecyclase

      • Serine-Threoninekinase - phosphorylate serine or threonine in the affected polypeptide

    cAMP

    Regulatory

    subunits

    Catalytic

    subunits

    cAMP response

    element binding

    (TGANNTCA)


    Transcription

    Eukaryotic Gene Regulation

    Tumor Necrosis

    Factor

    • Signal transduction

    • Example – Sequestration of inactive transcription factor

    TNF Receptor

    Associated Factor 2

    IKB kinase

    Inhibitor

    of KB

    Nuclear Factor

    kappa-light-chain-

    enhancer of

    activated B cells


    Transcription

    Eukaryotic Gene Regulation

    • Signal transduction


    Transcription

    The Cell Nucleus…

    • Post-transcriptional control

      • Alternative splicing – a single gene can encode two or more related proteins; multiple processing pathways for the transcript

        • Genes of complex plants & animals have numerous introns & exons —> use a different exon combination, get a different protein

        • Roughly 40 – 60% of human genes are subject to alternate splicing


    Transcription

    The Cell Nucleus…

    • Post-transcriptional control

    • Translational-level control

    • 3 aspects of translational-level control

      • A. Localization of mRNAs to certain sites within a cell

      • B. Controlling whether or not an mRNA is translated and, if so, how often

      • C. Controlling the half-life of the mRNA, a property that determines how long the message is translated

    • Mechanisms usually work via interactions between mRNAs & cytoplasmic proteins


    Transcription

    The Cell Nucleus…

    • Post-transcriptional control

      • mRNAs contain noncoding segments, called untranslated regions (UTRs) at both their 5' & 3' ends; these are sites where most translational control is effected

        • 1. 5' UTR extends from methylguanosine cap at start of message to AUG initiation codon

        • 2. 3' UTR extends from termination codon at end of coding region to the end of the poly(A) tail attached to nearly all eukaryotic mRNAs


    Transcription

    The Cell Nucleus…

    • Translational-level control…

    • Cytoplasmic localization of mRNAs

      • Example: the fruit fly, anterior-posterior axis

        • 1. Axis formation is influenced by localization of specific mRNAs along same axis in the oocyte

        • 2. Bicoid mRNAs preferentially localized at anterior end; oskar mRNAs preferentially localized at opposite end

        • 3. Protein encoded by bicoid mRNA is critical for head & thorax development; oskar protein is required for formation of germ cells, which develop at posterior end of larva

        • 4. Localizing mRNAs is more efficient than localizing their corresponding proteins, since each mRNA can be translated into large numbers of protein molecules


    Transcription

    The Cell Nucleus…

    • Translational-level control…

    • Cytoplasmic localization of mRNAs

      • 3' UTR governs localization of bicoid & oskar mRNAs

        • 1. Join foreign gene coding region to DNA sequence encoding 3’ UTR of oskar or bicoid

        • 2. Place in fruit flies & see what happens when the foreign gene is transcribed during oogenesis —> foreign gene goes to site determined by its 3’ UTR

        • 3. Localization of mRNAs is mediated by specific proteins that recognize mRNA localization sequences (zipcodes) in this region of mRNA


    Transcription

    The Cell Nucleus…

    • Translational-level control…

    • Controlling mRNA translation

    • Example: mRNAs stored in unfertilized egg are templates for proteins synthesized during the early stages of development;

      • rendered inactive by association with inhibitory proteins

      • Activation of these stored mRNAs involves at least two distinct events:

        • 1. Release of bound inhibitory proteins

        • 2. Increase in length of poly(A) tails by action of an enzyme residing in egg cytoplasm


    Transcription

    The Cell Nucleus…

    • Translational-level control…

    • Controlling mRNA stability

    • The longer an mRNA is present in cell, the more times it can serve as template for polypeptide synthesis

      • c-fos mRNA made in response to changes in external conditions in many cells; degraded rapidly in cell (half-life of 10 - 30 min); involved in cell division control

      • In contrast, dominant cell protein mRNAs in a particular cell, like those for hemoglobin, (half-life >24 hours)


    Transcription

    The Cell Nucleus…

    • Translational-level control…

    • Controlling mRNA stability

    • mRNA longevity is related to length of poly(A) tail

      • 1. Early study - mRNAs lacking poly(A) tails are rapidly degraded after injection into cell, whereas same mRNA with poly(A) tail is relatively stable

      • 2. Typical mRNA has ~200 adenosine residues when it leaves nucleus

      • 3. Gradually reduced in length as it is nibbled away by poly(A) ribonuclease

      • 4. No effect until the tail is reduced to ~30 A residues; once shortened to this length, the mRNA is usually degraded rapidly


    Transcription

    The Cell Nucleus…

    • Translational-level control…

    • Controlling mRNA stability

    • Tail length not the whole story;

      • mRNAs starting with same size tail have very different half-lives –

      • 3' UTR plays role

      • 3'-UTR of α-globin mRNA contains a number of CCUCC repeats that serve as binding sites for specific proteins that stabilize mRNA; if these sequences are mutated, the mRNA is destabilized

      • Short-lived mRNAs often contain destabilizing sequences (AU-rich elements; AUUUA repeats) in their 3' UTR; thought to bind proteins that destabilize mRNA


    Transcription

    The Cell Nucleus…

    • Post-translational control…

    • Controlling protein stability

    • Every protein is thought to have characteristic longevity (half-life) or the period of time during which it has a 50% likelihood of being destroyed

      • A. Some enzymes (those of glycolysis or erythrocyte globin molecules) are present for days to weeks

      • B. Other proteins required for a specific, fleeting activity (regulatory proteins that initiate DNA replication or trigger cell division) may survive only a few minutes

      • C. All of the proteins, regardless of expected survival time, are degraded by proteasomes

      • D. Factors controlling a protein's lifetime are not well understood


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