Lecture 4 - Genes and Development Flashcards by Ahn Studies

principles in gene development

genomic equivalence
selective gene expression

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all cells contain identical set of genes

genomic equivalence

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expts in genomic equivalence

Spemann
Briggs & King
Wolffian regeneration

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different cells activate different genes at different times

selective gene expression

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“__ __ __ from the same __ __”

differential gene expression
nuclear repertoire

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from DNA to chromosome

DNA
Nucleosome
Solenoid
Looped Chromosome
Condensed Chromosome
Mitotic Chromosome

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dna size

2nm

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nucleosome size

11nm

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solenoid size

30nm

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looped chromosome size

300nm

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condensed chromosome size

700nm

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mitotic chromosome size

1,400 nm

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10 year project
physically map the human genome

the human genome project

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duration of the human genome project

1988-2003

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human genome

3x10^9 bp)

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results of the human genome project:
total no. of genes

~30K

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results of the human genome project:
shortest gene

histone - 500 NT

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results of the human genome project:
largest gene

DMD (dystrophin gene) - 2,200kb

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results of the human genome project:
Chrom I

2,968 genes

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results of the human genome project:
Chrom Y

231 genes

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results of the human genome project:
Chrom 17

associated with diseases

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eukaryotic gene structure

promoter region
cap sequence or ACATTG
ATG codon
exons
introns
translation termination codon
3’ untranslated region

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binding site of RNA polymerase and subseqent initiation of transcription

promoter region

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transcription initiation stie
represents the 5’ end of RNA, which will receive a “cap” of modified nucleotide soon after it is transcribed
vary among genes; necessary for the binding of mRNA to ribosomes and its translation

cap sequence or ACATTG

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cap sequence or

ACATTG

why is cap sequence necessary

for the binding of mRNA to ribosomes and its translation

for initation of translation

ATG codon

- intervening sequence between initiation points of transcription & translation - determines the rate of translation initiation

leader sequence

base pairs coding for a protein

exons

- non-coding sequences interspersed among the exons - may be longer and more numerous than exons

introns

TAA

translation termination codon

TAA

- transcribed but not translated into protein - AATAA sequence where a "tail" of adenylate resudes are added - poly(A)tail confers stability and translatability on the mRNA

3' untranslated region

sequence in the 3' untranslated region

AATAA

how many adenylate residues are added in AATAA sequence

200-300

confers stability and translatability on the mRNA

poly (A) tail

Levels of control

1. differential gene transcription 2. selective RNA processing 3. selective mRNA translation 4. differential protein modification

levels of gene control

1. transcription 2. posttranscriptional

to become an active protein the RNA must be?

1. processes into mRNA (removal of introns) 2. translocated from nucleus to cytoplasm 3. translated by the protein-synthesizing apparatus 4. posttranslationally moified to become active

two types of regulatory elements

1. cis-regulators 2. trans-regulators

represent specific DNA seqence on a given chromosome which act only on adjacent genes

cis-regulators

dfiferent types of cis-regulators

1. promoters 2. enhancers

- required for the binding of RNA polymerase and accurate initiation of transcription - specify the times and places of transcription

promoters

- DNA sequence - activate the utilization of the promoter - control the efficiency and rate of transcript - functions by binding to transcription factors - activate only promoters on the same chromosomes

enhancers

promoter structure

- TATA box or Goldberg-Hogness box - about 30 bp

where is TATA box found

~30bp upstream from site where transcription begins

can be analyzed by determining which of its bases are necessary for efficient transcription

functional anatomy

promoter function

1. bind RNA polymerase 2. specify the places and times the transcription can occur from the gene

will not bind to naked DNA sequence

eukaryotic RNA polymerases

what do eukaryotic RNA polymerases require to bind efficiently to the promoter

protein factors

transcribes protein-coding genes

RNA pol II

how many protein have been shown necessary for the proper initation of transction by RNA pol II

proteins that have been shown necessary for the proper initation of transction by RNA pol II

basal transcription factors

recognizes the TATA box through one of its subunits

TFIID

subunit of TFIID

TATA-binding protein (TBP)

TFIID serves as the what?

- foundation of transcription initiation complex - serves to keep nucleosome from forming in this region

stabilizes TFIID

TFIIA

TFIID can bind to what after stabilizied by TFIIA

TFIIB

other transcription factors used to release RNA pol from complex

TFIIE, F, G

requirement for enhancers

1. DNA sequence that can activate the utilization of a promoter, controlling the efficiency and rate of transcription from that particular promoter 2. primary elements responsible for tissue-sepcific transcription 3. function by binding specific regulatory proteins called transcription factors

function of enhancers

regulate the temporal and tissue-specific expression of all differentially regulated genes

importance of enhancers

1. required by genes for their transcription 2. major determinant of differential transcription in space and time

the combination of transcription factors that causes particular genes to be transcribed

modular

the same transcription factors that activate the transcription of one gene can be used to what?

repress the transcription of other genes

repress tha transcription of other genes

negative enhancers = silencers

- soluble molecules from one gene and interact with genes on the same or different chromosomes - with sequence specific DNA-binding domain

trans-regulators

trans-regulators have sequence specific __ __ __

DNA-binding domain

enables that transcription factor to interact with proteins involved in binding RNA polymerase

trans-activating domain

- proteins that bind to enhancer or promoter reigons and interact to activate or repress the transcription of a particular gene - can bind to specific DNA sequences - can be grouped together in families based on similarities in structure

transcription factors

share a common framework structure in their DNA-binding sites, and slight differences in the amino acids at the binding site can alter the sequence of the DNA to whcih the factor binds

transcription factors

Three major domains of transcription factors

1. DNA-binding domain 2. trans-activating domain 3. protein-protein interaction domain

recognizes a particular DNA sequence

DNA-bingind domain

- activates or suppresses the transcription of the gene whose promoter or enhancer it has bound - enables the transcription factor to interact with proteins involved in binding RNA polymerase

trans-activating domain

allows the transcription factor's activity to be modulated by TAFs or other transcription factors

protein-protein interaction domain

- are sequences tat act specifically to block transcription - useful in restricting the transcription of a particular gene to a particular geoup of cells or regulating the timing of the gene's expression

silencer

ex of silencer

when the endodermal tube contacts with the cardiac mesoderm, the heart precursors are able to instruct the endodermal tube to begin forming the liver and to start transcribing liver-specific genes

Trans-regulatory factors

1. homeodomain proteins 2. zing finger standard 3. basic helix-loop helix TF 4. basic leucine zipper 5. nuclear hormone receptors 6. Sox-2 TF

- critical for specifying the anterior-posterior body axes - 60aa arranged in a helix-turn-helix, such that the third helix extends into the major groove of the DNA it recognizes

homeodomain proteins

major groove of Hox tf

helix 3

minor groove of Hox TF

NH2

mutation transform body segment to another

homeosis

region that comprises the homeodomain and then 2nd DNA-binding region

POU domain

- has two or more "DNA-binding fingers" helical domains - coordinatied by 2 cysteins and 2 histidines

zinc finger standard

ex. of zinc finger

WT Krox 20

critical for kidney and gonads development

for hindbrain development

Krox20

- binds to DNA via a region of basic AA (10-13 res) - may form a dimer with positive or negative regulators

basic helix-loop helix TF (bHLH)

ex. of basic helix-loop helix TF

1. E12 2. E47

alpha helix with several luecine reside that bind with other bZip proteins

basic leuzine zipper (bZip)

mediate the effect of hormones on genes

nuclear hormone receptors

bends DNA from "I" to "L"

Sry-Sox

important in mammalian primary sex determination

Sox-2 TF

types of RNA polymerase

1. RNA Pol I 2. RNA Pol II 3. RNA Pol III

transcribes large ribosomal RNAs

RNA pol I

crucial to cell differentiation - determine which genes transcribed and produced

transcription of a gene

post-transcriptional processes

1. capping 2. polyadenylation 3. splicing

addition of 7-methyl-guanylate to the 5' end of pre-mRNA

capping

what is added during capping

7-methyl-guanylate

- a chain of 150-200 adenylate nucleotide is attached to the 3' end of the pre-mRNA after transcription - stabilize the mRNA and alloows its exit and translation

polyadenylation

how many adenylate nucleotide is added during polyadenylation

150-200 adenylate

removal of non-coding sequences from pre-mRNA to produce the mature mRNA

splicing

acts on the gene

epigenetic mechanism

chemical modifications

1. methylation 2. acetylation

can inactivate genes

methylation of DNA

allows DNA unpacking and transcription

acetylation of histones

- might change the structure of the gene thus regulation its activity - stabilizies nucleosomes and prevents transcription factors from binding

DNA methylation

three areas in which DNA methylation can contribute to differential gene activity

1. methylation of promoter 2. responsible for distinguishing certain egg-derived and sperm-derived genes in mammals 3. continued repression of the genes on one of the two X chromosomes in each female mammalian cell

temporal and spatial regulation on genes encoding tissue-specific proteins

methylation of promoter

only one will be expressed during early development

distinguishing between certain egg/sperm-derived genes

where does methylation occur exclusively

lysine 9 of histone 3 (H3-K9)

what does methylation promote binding of

HP1 proteins (heterochromatin proteins)

condensed and replicates after most of the chromatin

heterochromatin

genes are active only if derived from paternal

gene imprinting

- removal of acetyl group - H4 lysine 16

deacetylation

H4 lysine 16

removes the positive charge from histone reducing the force of attraction with DNA leading to wider opening of the chromatin

acetylation of lysine

- restores the positive charges and promotes close attraction between histone and DNA - condensed chromosome

deacetylation

200 or so adenyl groups added to the 3' end

polyadenylation

initiates transcription producing 20-25 NT

CTD (carboxyl terminal domain)

% of genes repressed in a cell

90%

% of genes expressed in a cell

10%

control of gene expression before transcription

1. selective gene amplification 2. gene rearrangement 3. chemical modifications

posttranscriptional-selective RNA processing

1. censorship 2. hnRNA splicing

which nuclear transcripts are processed into cytoplasmic messages "chosen few"

censorship

the same nuclear RNA is spliced into different mRNAs

hnRNA splicing

% of human genes that are alternatively spliced

75%

- process results in down regulation of a gene at the RNA level (after transcription) - there is also gene silencing at the transcriptional level

post-transcriptional gene silencing (PTGS)

other term for post-transcriptional gene silencing (PTGS)

RNA interference / RNAi

gene silencing at transcriptional level

1. transposons 2. retroviral genes 3. heterochromatin

splicesosome complex

splicing regular protein+ snRNPS & SF joined

joins exons

ligase

important source of protein diversity

alternative gene splicing

different types of alternative RNA splicing

1. alternative 5' splicing 2. alternative 3' splicing 3. intron retention 4. mutually exclusive exons 5. exon skipping

part of an exon may be included in the splicing at 5' end

alternative 5' splicing

part of an exon may be included in the splicing at 3' end

alternative 3' splicing

an intron is included in the final mRNA

intron retention

% of human genes with intron retention

2-5%

different exons found in 2 diferent mRNAs

mutually exclusive exons

2x bigger than ordinary baby zebra

zebroid foal

- the longer an mRNA persists, the more protein can be translated from it - stability of a message - length of its poly(A) tail depends upon sequences in the 3' untranslated region

differential mRNA longevity

polyadenylation confers stability

increase rate of translation

affects longevity of casein during lactation

prolactin

- stored oocyte mRNA - informations are dormant until translated at/near fertilization - activated by ionic signals at fertilization/ovulation - other stored messages encode proteins that determine the fate of cells

selective inhibition of mRNA translation

provide positional information in the Drosophila embryo

bicoid and nanos

- time of mRNA translation regulated, but so is the place of RNA expression - after fertilization, it is found only in vegetal blastomeres

control of RNA expression by cytoplasmic localization

post-translational modifications

1. phosphorylation 2. lipidation 3. ubiquitination 4. disulfide bond 5. acetylation 6. glycosylation

adds a phosphate to serine, threonine or tyrosine

phosphorylation

attaches a lipid, such as a fatty acid, to a protein chain

lipidation

adds ubitquitin to a lysine reside of a target protein marking it for destruction

ubiquitination

covalently links the 5' atoms of two different cysteine residues

disulfide bond

adds an acetyl group to the N-terminus of a protein to increase stability

acetylation

attaches a sugar, usually to an "N" or "O" atom in an amino acid side chain

glycosylation

how do cells become different

selective gene expression

when does contrl of gene expression occur

at different levels from transcription to translation

initates and maintain specific gene expression

particular combination of gene regulatory proteins (TF)

gene control is a __, not a linear diagram

maze

gene is not an indepent entity controlling the synthesis of protein:

it both directs and is directed by protein synthesis