Gene Regulation, Part 2 Lecture 15 (cont.) Fall 2008

Eukaryotic Gene Expression Regulation occurs at many levels 1 Fig. 18.6

Chromatin Packing Chromatin –Complex of DNA and proteins Histones –Small proteins –Positive charged so bind with DNA Nucleosomes (10 nm fiber) –DNA wound twice around 8 histones –Linker DNA: DNA between the nucleosomes –Histones release from DNA during DNA replication and gene expression 2 Fig

Chromatin Packing 30 nm fiber –Interactions between histone tails and linker DNA Looped domains (300 nm fiber) –Loops form around a protein scaffold Metaphase chromosome (700 nm) –Further compaction occurs –Packing steps highly specific 3 Fig

Chromatin Packing Chromatin much less condensed in Interphase –Nucleosomes, 30 nm and some loops (but no scaffolding Loops attached to nuclear lamina –May help organize regions of chromatin where genes active Heterochromatin –Areas of highly condensed chromatin during interphase E.g., centromeres, telomeres Euchromatin –Less condensed regions Read Fig Inquiry 4

Chromatin Packing & Gene Regulation Transcription affected by: –Where promoter located in relation to nucleosomes –Genes within heterochrome typically not transcribed –Chemical modifications to histones and DNA E.g., X chromosome inactivation Fig

Chromatin Packing & Gene Regulation Histone acetylation –Addition of acetyl group (-COCH 3 ) to histone tail –Neutralizes positive charge of tail No binding to neighboring nucleosomes Genes available for transcription –Requires enzymes May be part of transcription factors binding to promoter Deacetylation 6 Fig. 18.7

Chromatin Packing & Gene Regulation Histone methylation –Adds methyl group (-CH 3 ) to histone tails –Promotes chromatin condensation Histone phosphorylation –Promotes unfolding of chromatin Many signals may work together 7 Fig. 18.7

Chromatin Packing & Gene Regulation 8 DNA methylation –Addition of methyl group to base (~cytosine) Inactivated X chromosome highly methylated Long term inactivation of genes Multiple mechanisms may work together –Certain proteins binding to methyl groups recruit histone deacetylation enzymes Both act to increase condensation of chromatin

Chromatin Packing & Gene Regulation Methylation patterns passed on in cell division –Genomic imprinting Variations in phenotype depending on whether an allele is inherited from the male or female parent –One allele “silenced” via methylation 9 Fig

Chromatin Packing & Gene Regulation Epigenetic inheritance –Modifications to chromatin that do not involve a change in DNA sequence –Inherited –Can be reversed 10

Regulation of Transcription Initiation Control elements –Segments of noncoding DNA that help regulate transcription by binding certain proteins 11 Fig. 18.8

Regulation of Transcription Initiation Transcription Factors –Mediate binding of RNA polymerase to promoter & initiation (eukaryotes) –One TF binds to TATA box Transcription initiation complex –Complex of RNA polymerase and transcription factors Fig

Regulation of Transcription Initiation General transcription factors –Essential for the transcription of all protein-coding genes –Most transcription factors bind to other proteins –Formation of transcription initiation complex allows for initiation and transcription Rate of transcription low 13

Regulation of Transcription Initiation Specific transcription factors –Control elements that control transcription of specific genes Increase or decrease rate of gene expression –Proximal control elements –Close to promoter –Distal control elements (Enhancers) More distant from promoter May be multiple enhancers per gene 14 Fig. 18.8

Regulation of Transcription Initiation Activators or repressors can bind to enhancers Activators bind to enhancers DNA bent to bring distal control element closer to promoter –DNA bending protein 15 Fig. 18.9

Regulation of Transcription Initiation Activators interact with mediator proteins Mediator proteins interact with general transcription factors Allows RNA polymerase to bind and promotes increased transcription 16 Fig. 18.9

Regulation of Transcription Initiation Repressors can inhibit gene expression –Block enhancer and prevent activator from binding –Block activator from binding with DNA –Recruit proteins that deacetylate histones 17

Regulation of Transcription Initiation Enhancers ~ 10 control elements –Control elements bind to only 1 or 2 specific transcription factors Combination of control elements important for differential gene expression Transcription activated only when particular combination of activator proteins available 18 Fig

Post-Transcriptional Regulation Alternative RNA splicing –Different RNA molecules produced from same primary transcript –Regulatory proteins control intron-exon choices 19 Fig

Post-Transcriptional Regulation mRNA degradation –Enzymes degrade mRNA after translation Bacterial mRNA – quick (few minutes) Eukaryote mRNA varies (hours to weeks) –Poly-A tail shortened by enzymes –Triggers removal of 5’ cap –Nuclease enzymes degrade mRNA 20

Post-Transcriptional Regulation Initiation of Translation Regulatory proteins bind to untranslated regions (UTR) and block binding of mRNA to ribosome Size of poly-A tail may prevent translation –Addition of adenine prompts translation “Global” activation of translation –Activates translation in egg after fertilization –Trigger of activation of translation initiation factors –E.g., light triggers in plants 21 Fig Fig

Post-Transcriptional Regulation Protein processing –Cleavage, phosphorylation, addition of sugars Protein degradation –Ubiquitin Marks protein for degradation –Proteasome Protein complex that unfolds & degrades proteins 22 Fig

Noncoding RNA & Gene Regulation ~ 1.5% of DNA codes for proteins Small portion of genome codes for small RNAs –E.g. tRNA, rRNA Large amount of genome transcribed into non- protein-coding RNA 23

Noncoding RNA & Gene Regulation microRNA (miRNA) Small, single stranded RNA –~20 nucleotides long Bind to complementary sequence in mRNA Degrades or blocks translation of target mRNA 24 Fig

Noncoding RNA & Gene Regulation Small interfering RNAs (siRNAs) –Similar effect as microRNAs –Involved in formation of heterochromatin at centromeres 25