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Prokaryotic expression

prokaryotic gene expression differs in many ways from eukaryotic differences in polymerases transcription factors do not exist in bacteria initiation of translation can differ
eukaryotes small ribosomal subunit binds the cap prokaryotes binds a specific sequence at the 5 end initiation factors differ

prokaryotics lack a nucleus and organelles

genes can be transcribed and translated AT THE SAME TIME! eukaryotes can process their RNA in the nucleus the organelle organization of the eukaryote cells means these cells can target specific proteins to specific organelles

What is a gene?
Mendel genetic information that gives rise to a specific phenotype Morgan assigned genes to specific chromosomes (loci) sequence of DNA nucleotides within the chromosome codes for a specific polypeptide sequence through transcription into mRNA and translation
but can also be transcribed into rRNA, tRNA, snRNA (non translated RNAs)

comprised of numerous regions

coding region = polypeptide sequence
introns and exons

untranslated regions surround the coding region

are transcribed into mRNA not translated by the ribosome

promoter upstream of the coding region not transcribed enhancer regions repressor regions

A gene: the final definition

a gene is a region of DNA that can be expressed to produce a final functional product that is either a polypeptide or an RNA molecule

Regulation of Gene Expression

bacterial operons allows the bacteria to regulate its transcription/translation machinery using the environment
operons participate in metabolic control turned on or off based on the metabolic requirements of the bacteria

operon: grouping of genes producing enzymes involved in a metabolic pathway

e.g. trpoperon, lacoperon promoter controls transcription of multiple downstream genes that code for enzymes that synthesize a specific metabolic component (e.g. amino acid) these downstream genes are called a transcription unit all downstream genes are transcribed when the promoter is activated coordinate control also contains an operator region on/off switch that ultimately controls the promoter and the downstream genes

The trp operon

codes for enzymes that will synthesize tryptophan operon is usually ON unless repressed comprised of a promoter and regulatory gene = tprR
upstream of the transcription unit another promoter region, an operator region and the genes coding for enzymes that synthesize tryptophan

the transcription unit is downstream of this region in the absence of tryptophan

the promoter promotes transcription of trpR translated into an inactive repressor protein allows the binding of the RNA polymerase to the transcription units promoter transcription of the trp genes translation and production of active enzymes = make tryptophan tryptophan physically binds the trpR protein and converts it into an active repressor protein the active trpR protein binds to the operator region of the operon prevent the binding of the RNA polymerase represses the transcription of the downstream metabolic enzyme genes no tryptophan made so the accumulation of tryptophan in the bacteria s environment inhibits its own tryptophan metabolic machinery CALLED A REPRESSIBLE OPERON tryptophan is the repressor

in the presence of tryptophan

The lac operon

codes for enzymes that processes lactose operon is usually OFF unless stimulated comprised of a promoter and regulatory gene = lacI
upstream of the transcription unit lacZ = b-galactosidase breaks lactose into galactose and glucose the promoter promotes transcription of lacI translated into an active repressor protein the active lacI protein binds to the operator region of the lacoperon prevent the binding of the RNA polymerase represses the transcription of the downstream metabolic enzyme genes no lactose processing enzyme made lactose physically binds the lacI protein and converts it into an inactive repressor protein the active lacI protein binds to the operator region of the operon promotes the binding of the RNA polymerase activates the transcription of the downstream metabolic enzyme genes processing of lactose into galactose and glucose so the accumulation of tryptophan in the bacteria s environment inhibits its own tryptophan metabolic machinery CALLED A INDUCIBLE OPERON lactose is the inducer

three downstream genes lacZ, lacY and lacA in the absence of lactose

in the presence of lactose

Positive gene expression

the trp and lac operons are examples of negative gene regulation
operons are switched OFF by a repressor

positive gene regulation

lacoperon also bacteria would prefer to use glucose rather than processing lactose when glucose is scarce/lactose is abundant
accumulation of a chemical cAMP (ATP derivative) cAMP binds a protein called CAP catabolite activator protein CAP is an activator better than lactose binds to the lacoperon promoter transcription PLUS lactose binds the lacI repressor inactivating its repressor function so transcription is positively controlled by the CAP protein rather than through the inactivation of the lacI repressor results in a higher level of lactose processing than that controlled by lactose levels alone

when glucose is abundant/lactose is abundant

low amounts of CAP can t promote the binding of the RNA polymerase lactose binds its repressor can t bind the promoter so RNA polymerase still binds but at a lower affinity than when CAP is involved production of lactose processing enzymes

Regulation of eukaryotic gene expression

typical human cell only expresses about 20% of its total genes in muscles and nerves even smaller fraction differences between cell types = differential gene expression 1.chromatin structure 2. regulation of transcription initiation 3. post-transcriptional regulation

Chromatin Structure
packing of DNA into histones can regulate its ability to be transcribed
closer the promoter is to the nucleosome less likely it will be transcribed attachment of the chromatin to the nuclear matrix (nuclear lamina) or the scaffolding of a chromosome can affects its transcription

genes within heterochromatin are usually not expressed chemical modifications of histones can modify the packing of the DNA
tails of the histones project outward modifications:
acetylation acetyl groups (COCH3) attached to the lysines of the tails
deacetylation is their removal- e.g. HDAC (histonedeacetylase) acetylation neutralizes the histones positive charge no longer bind to the neighbouringhistone loosens the packing and allows access to the transcription machinery acetylation = gene expression

methylation methyl groups (CH3) attached to the tails promotes condensation of chromatin
direct methylation of the DNA bases can also occur usually cytosine can inactivate the gene i.e. not transcribed longer-term methylation/shut-down is important for development of the embryo methylation retained over mitosis daughter DNA is methylated just like the parent so the pattern of methylation can be passed on methylation patterns can also develop de novo DNA traits produced by things other than the DNA sequence = epigenetic inheritence can be reversed in succeeding generations = ???? mechanism????

phosphorylation can decondense the chromatin

Transcriptional initiation
after the chromatin structure is changed to allow transcription regulate the initiation of transcription 1st level promote the binding of the RNA polymerase to the promoter
upstream of the 1stexon site for RNA polymerase II binding assisted through the binding of other proteins = transcription factors some transcription factors regulate transcription of ALL genes = general transcription factors
some binding the DNA directly sequence called the TATA box others bind RNA polymerase II others bind other transcription factors e.g. CBFA-1 TF for osteogenic gene expression

others are specific give a higher rate of transcription

additional levels control elements bound by the specific transcription factors

elements near the promoter = proximal control elements
some biologists consider these part of the promoter can be bound by TFs that act as activators or repressors

away from the promoter = distal control elements or enhancers

general TFs, specific TFs, RNA pol II and other transcriptional mediators come together to form a large protein complex near the promoter = transcriptional initiator complex activator TFs have two things in common
1. DNA binding domain 2. Activation domain(s) bind other TFs or mediator proteins can bind directly to the control elements blocking the binding of activator TFs turn off transcription even when the activator TF is bound block the formation of the initiator complex

repressor TFs

Cell type specific gene expression

the different sequences of the control elements are quite small
~12 control element sequence in the eukaryotic cell

so it isn t the sequence of these elements that is important it is the combination of the control elements in the gene that is it is also the TFs they bind
many cell types can express the same TFs not all will be active

these combinations can differ from cell type to cell type

allows for the transcription of albumin in liver cells and the crystallin gene in cells of the lens (even though both genes are in the genome of both cell types)

Coordinate gene expression

how do eukaryotic cells turn related genes on or off at the same time? in bacteria cluster them into an operon one way cluster the related genes in the same region of a chromosome
changes in the chromatin structure allows the coordinate activation of their promoters

not always the case multiple chromosomes

have a similar combination of control elements steroid hormones act this way
e.g. vitamin D binds a region of DNA = vitamin D response element on any gene/chromosome that bears it

Post-transcriptional regulation
occurs after transcription 1. RNA processing
transcription of DNA within the gene -> primary RNA transcript (Figure 8.8) RNA is processed into mRNA with its coding region, its 5 and 3 UTRs
5 cap and poly A tail added on introns are spliced out how the splicing occurs can give multiple mRNA transcripts spliced regions determined by regulatory proteins cell-type specific

2. mRNA degradation
life span of mRNA within the cell determines its availability for translation
varies from mRNA to mRNA e.g. Hb mRNA = 120 days!!

initiation of breakdown starts with the removal of the polyA tail then triggers the removal of the 5 cap nucleases then chew up the remaining RNA degradation can be sped up by the binding of microRNA sequences (miRNA) to their complement in the mRNA
blocks the translation of the targeted mRNA and promotes its breakdown

3. translation initiation
binding of regulatory proteins to the untranslated regions of the mRNA prevents binding of ribosome lack of a 5 cap lack of a poly A tail can block translation also important for ribosome binding

Post-transcriptional regulation: siRNA

RNA can inhibit translation small interfering RNAs = siRNA produced when an enzyme (Dicer) chops up a primary RNA transcript small pieces of double-stranded RNA formed figure 18.13 siRNA binds onto to its mRNA complement and prevents its translation

Post-translation regulation
protein processing & degradation degradation rate = half-life of the protein
attachment of a small protein called ubiquitin these proteins are targeted for degradation by the proteosome(giant protein complexes within the cell)

modifications essential for function occur in the ER and Golgi apparatus ER modifications:
1. formation of disulfide bonds
help stabilize the tertiary and quaternary structure of proteins misfolded proteins remain in the cytoplasm and are degraded only properly folded proteins get transported to the Golgi for additional processing and transport

2. folding of the peptide chain 3. addition and processing of carbohydrates (glycosylation) 4. breakage of specific peptide bonds proteolytic cleavage 5. assembly into multimeric proteins (more than one chain)

Golgi modifications:
1. glycosylation addition of chains of carbohydrates to specific amino acids 2. trimming of the protein

Gene expression & development

embryonic development requires the coordinated expression of many genes PLUS the differential expression of others these events lead to the specialization of stem cells into the many types of the embryo = cell differentiation these cells are organized into tissues at specific locations giving the organism shape = morphogenesis how does this arise from ONE fertilized cell?? materials in the egg from the mother set up a program of gene regulation carried out at the start of development first differences in cell types determined by the cytoplasm and inductive signals
cytoplasmic determinants source of information during the earliest cell divisions Figure 18.15
contents of the egg are NOT homogenous mRNA, proteins and organelles are unevenly distributed throughout the egg influences the earliest stages of embryonic development different materials are partitioned differently into the daughter cells as division occurs

inductive signals signals sent by nearby cells (paracrine signaling)

binds onto receptors and influences the target cells behavior called signal transduction inducible signals induce an event in the cell

Cell Differentiation & Gene Expression

embryonic development requires cell specialization = differentiation first stages commit the cell to its fate = determination or commitment commitment is followed by differentiation what results is a differentiated cell making cell type and tissue typespecific proteins first events are genetic/molecular in level- result in commitment
new genes get expressed and new proteins appear many are new transcription factors these regulate additional genes sets of genes regulated in a sequential manner e.g. bone differentiation
Cbfa-1 = master transcription factor for bone development
binds to the control elements of numerous proteins involved in the differentiation of osteoblasts controls the expression of many osteogenic genes

e.g. muscle differentiation

isolated specific genes and expressed them in undifferentiated precursors watched to see what would happen would the precursor differentiate and how? figured out genes involved in the determination of muscle cells and their continued differentiation MyoD = master transcription factor for muscle development
binds control elements of many muscle differentiation genes and other muscle transcription factors can even induce myogenic differentiation in non-muscle precursors like stem cells, adipose cells and liver cells

Pattern formation and the body plan

cytoplasmic determinants and inducible factors not only start the process of determination and differentiation but help organize the developing cells into a body plan called pattern formation first events - establishment of the axes of the embryo
front/back, left/right, head/tail etc known as positional information tells the differentiating cells its position in the body and helps determine its future differentiation result

pattern formation studied by genetically manipulating the fruit fly and observing the resulting progeny
pioneered by Ed Lewis in the 1940s mapped mutations to the genome and altered the genome to make more mutants discovered what are called homeobox genes (or homeotic genes) the genes of pattern determination many genes named after their mutations e.g. wingless, hairy some have very bizarre names e.g. sonic hedgehog, patched, jagged, bicoid
first determined by cytoplasmic factors encoded by maternal effect genes = a gene that when mutated in the mother results in a mutated offspring despite the offspring s genome these gene products are placed in the egg prior to fertilization in the ovary the maternal effect genes controlling orientation of the egg = egg-polarity genes e.g. bicoid= two tailed mutation in this gene results in a fly that lacks the front half of its body posterior structures at both ends!!! found that the product of bicoid concentrates at the site that will become the anterior half of the body = morphogen gradient morphogen gradient = chemicals that help determine the form of the embryo figure 18.19 decreasing amounts of bicoid protein from anterior to posterior end of embryo

axis establishment and homebox genes