Chapter 16

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Mechanisms of transcriptional activator function

Activators are transcription factors that bind to enhancers and other activating sequences. •Activators are responsible for much of the variation in levels of transcription of different genes. •Increase levels of transcription by interacting directly or indirectly with basal factors at the promoter.

General transcription factors bind to promoters

Ordered pathway of assembly at promoter: 1. TBP (TATA-box binding protein) binds to TATA box 2. TAFs (TBP-associated factors) bind to TBP 3. RNA pol II binds to TAFs

DNA methylation

Transcription can be regulated by DNA methylation A methyl group (-CH3) is added to the cytosine base in a 5' CpG3' dinucleotide by DNA methyl transferases (DNMTs)

Chromatin immunoprecipitation-sequencing (ChIP-Seq)

Used to identify locations bound by transcription factors and histones

Gene expression profiling with RNA-seq

Used to quantitatively measure when and where a gene is expressed

Dimerization domain

allows proteins to form dimers.

How can enhancers be identified and how do they function?

•Constructing a recombinant DNA molecule that has a putative enhancer sequence fused to a reporter gene such as the green fluorescent protein (GFP) •Generating a transgenic organism that has the recombinant DNA in its genome

enhancers

-DNA sequence that can be far away from gene -Augment the basal level of transcription -May be located either 5' or 3' to the transcription start site •Still function when moved to different positions relative to promoter

promoters

-DNA sequence that is usually directly adjacent to the gene -Is always very close to the gene's protein-coding region -Bind RNA polymerase -Often have TATA box (initiation box) -Allow basal level of transcription

RNA polymerase in eukaryotes

-Different polymerases RNA Pol I: rRNA RNA Pol II: mRNA(all protein-coding genes) and miRNA(micro-RNA) RNA Pol III: tRNA

Prokaryotic transcriptional regulation

-No nucleus. Transcription and translation take place in same cellular compartments; often coupled -Genes not divided into exons and introns -One RNA polymerase -Promoters not wound up in chromatin

eukaryotic transcriptional regulation

-Transcription takes place in nucleus; translation in the cytoplasm -Exons separated by introns, which are taken out by posttranscriptional splicing -Enhancers are far from promoter and often needed for transcription initiation -Promoters need to be cleared of chromatin to allow access to RNA polymerase -Gene regulation controls cellular differentiation into hundreds of specialized cell types

insulators

Insulators: DNA sites that bind proteins in order to organize chromosomes into loops; an enhancer can interact only with promoters in the same loop Insulators are sequences located between an enhancer and a promoter They block access to the promoter

Enhancers can be identified by:

Constructing a recombinant DNA molecule that has a putative enhancer sequence fused to a reporter gene such as the green fluorescent protein (GFP) Generating a transgenic organism that has the recombinant DNA in its genome If DNA fragment contains an enhancer, then the GFP will be expressed

DNA methylation is stably maintained

DNMT works on hemimethylatedDNA (DNA methylated on one of the two strands)

How do miRNAs regulate gene expression?

Inhibition of mRNA translation•Degradation of target mRNA

Mechanisms of repressor function

Repressor proteins can act through competition with an activator protein. •An indirect repressor interferes with the function of an activator. •Competition due to overlapping binding sites. •Repressor binds to activation domain (quenching) .•Binding to activator and keeping it in cytoplasm. •Binding to activator and preventing homodimerization.

Mechanisms of repressor function

Repressor proteins suppress transcription initiation by recruiting corepressors •Corepressors can have two alternate functions:-Prevent RNA pol II complex from binding the promoter-Modify histones with repressive marks (H3K27me3, H3K9me3)

Transcription factors

Sequence-specific DNA binding proteins •Bind to promoters and enhancers •Recruit other proteins to influence transcription •Three types: general factors, activators, repressors

insulators

are sequences located between an enhancer and a promoter•They block access to the promoter organize genomic DNA into loops -Enhancers activate promoters located in the same loop

DNA binding domain

binds to DNA sequence motif.

Activation domain

binds to other proteins (basal factors or coactivators).

Recruit coactivators to

increase chromatin accessibility.

Post-transcriptional regulation by microRNAs (miRNAs)

miRNAs are transcribed by RNA polymerase II from non-coding regions or introns of protein-coding genes.•The primary transcripts have double-stranded stem loops.•Droshaexcises stem-loop from primary miRNA (pri-miRNA) to generate pre-miRNA of ~ 70 nt•Dicer processes pre-miRNA to a mature duplex miRNA (~ 21-22 nt)•One strand is incorporated into RNA-induced silencing complex (RISC) •Mature miRNAs base-pair partially to their target mRNAs and downregulate them.

Paternally imprinted

paternal allele is methylated and silenced

Genomic imprinting

phenomenon in which expression of an allele depends on the parent that transmits it. •Methylation at imprinting control regions (ICRs) silences gene expression •RNA-seq has identified about 100 imprinted genes


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