Molecular genetics & gene expression Mat Halter and Neal Stewart 2016

Discussion questions What are the differences between DNA and RNA? Describe the main parts of a gene and its functions. What role do cis-regulatory elements and trans-acting factors play in gene regulation? What is responsible for the wide diversity of protein structure found in nature? In what different ways can gene expression be regulated?

A chromosome is composed of nucleosome-bound DNA called chromatin

Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid April 25, 1953 Francis CrickJames Watson Figure 6.2

A pair with T G pair with C Nucleotide base pairing occurs through “hydrogen bonding” Strands have directionality from 5’ to 3’ and when paired strands are in “antiparallel” orientation Nucleotide base pairing

DNA and RNA are structurally similar

Some differences of RNA and DNA

Difference between thymine and uracil

Discussion questions What are the differences between DNA and RNA? Describe the main parts of a gene and their functions. What role do cis-regulatory elements and trans-acting factors play in gene regulation? What is responsible for the wide diversity of protein structure found in nature? In what different ways can gene expression be regulated?

DNARNAProtein Transcription The Central Dogma

Figure 6.5 Eukaryotic gene structure

Transcription of DNA into mRNA TF AAAAA RNA polymerase II TF mRNA = messenger RNA

Fig 6.6

Eukaryotic genes contain introns which are spliced to form mature mRNA

Polycistronic prokaryotic message = several mRNAs are regulated by one promoter Monocistronic eukaryotic message mRNA structure differs between prokaryotes and eukaryotes

Discussion questions What are the differences between DNA and RNA? Describe the main parts of a gene and their functions. What role do cis-regulatory elements and trans-acting factors play in gene regulation? What is responsible for the wide diversity of protein structure found in nature? In what different ways can gene expression be regulated?

Cis- acting regulatory elements Promoter region- Located immediately upstream of the transcription start site and serves as a binding site for the RNA -polymerase II complex. Enhancer region- Promotes transcription by recruiting histone- modifying enzymes that open the chromatin structure, making the sequence more accessible—sometimes acts in trans. Insulators- Sequences that have the ability to protect genes from inappropriate signals emanating from their surrounding genome. Vascular Tissue- Specific Promoter GUS gene Terminator

Transcription factors: proteins that bind to promoters to affect transcription Transcriptional activators- Recruits the RNA polymerase complex to the transcription start site by binding to either sequences in the promoter or distant cis-acting elements to increase transcription. Transcriptional repressor- Prevents transcription of a gene by interfering with RNA polymerase activity on a promoter RNA Polymerase PromoterLacZLacYLacA mRNA VIDEO

Transcription of DNA into mRNA TF AAAAA RNA polymerase II TF mRNA = messenger RNA The TFs in this case are transcriptional activators

Discussion questions What are the differences between DNA and RNA? Describe the main parts of a gene and their functions. What role do cis-regulatory elements and trans-acting factors play in gene regulation? What is responsible for the wide diversity of protein structure found in nature? In what different ways can gene expression be regulated?

h?v=D3fOXt4MrOM Transcription and translation, the movie.

Translation DNARNAProtein The Central Dogma of Molecular Biology

The central dogma revisited The order of the DNA template or coding strand is 3’ to 5’ This determines the order of the mRNA strand (5’ to 3’) because DNA template is complementary to the mRNA strand.

Transfer RNA (tRNA) Amino Acid Amino Acid

AUG UCG ACA GGC UGA

Initiation of translation

The start codon is found by scanning downstream from the 5’ end of the mRNA VIDEO

Amino acid backbone

20 amino acids

Amino AcidsThree-Letter AbbreviationOne-Letter Abbreviation AlanineAlaA ArginineArgR AsparagineAsnN AspartateAspD CysteineCysC GlutamineGlnQ GlutamateGluE GlycineGlyG HistidineHisH IsoleucineIleI LeucineLeuL LysineLysK MethionineMetM PhenylalaninePheF ProlineProP SerineSerS ThreonineThrT TryptophanTryW TyrosineTyrY ValineValV TABLE 6.1 The 20 Amino Acids Commonly Found in Proteins

Figure 6.11 Figure 6.11 Polypeptide structure. The building block of a polypeptide is the peptide bond formed between amino acids. Peptide bonds connect amino acids to create a polypeptide chain. Proteins are formed through the association of individual polypeptide chains that may be identical to each other or unique in sequence.

Figure 6.12 Figure 6.12 The genetic code gives rise to either overlapping or non-overlapping reading sequences. A codon consists of three consecutive nucleotides that code for an amino acid. The nucleotides in a codon may give rise to multiple amino acids depending on the reading frame.

Discussion questions What are the differences between DNA and RNA? Describe the main parts of a gene and their functions. What role do cis-regulatory elements and trans-acting factors play in gene regulation? What is responsible for the wide diversity of protein structure found in nature? In what different ways can gene expression be regulated?

Gene expression regulation Transcriptional level: –Transcription factors (environmental, physiological) –Alternate splicing Translational level: –Elongation factors –RNA interference

Discussion questions What are the differences between DNA and RNA? Describe the main parts of a gene and their functions. What role do cis-regulatory elements and trans-acting factors play in gene regulation? What is responsible for the wide diversity of protein structure found in nature? In what different ways can gene expression be regulated?

Focus questions How important are cis-regulatory elements and trans-acting factors in gene regulation? What are the control points that can regulate gene expression?

Transcription revisited

Promoter elements not required for transcription initiation CAAT box – usually located at -70 to -80 within the promoter GC box Other gene-specific elements (light- responsive, nutrient-responsive, etc.) Enhancer elements

What are some biological roles of transcription factors? Basal transcription regulation – general transcription factors Development Response to intercellular signals Response to environment Cell cycle control

The CRT/DRE response element responds to dehydration and cold-induced transcription factors (CBF)

Figure 6.7 Transcription factors

Enhancer can work from downstream and upstream region

Enhancers Their location is not fixed. Location could be in the upstream or downstream DNA, in intron, exon or in the untranslated region. They enhance transcription by acting on promoter in cis (typically) Each enhancer has its own binding protein. These proteins are trans-regulatory activating factors Sequence of enhancers is variable. Enhancers regulate tissue-specific and temporal expression of genes.

DNA-binding domains allow transcription factors to bind directly to a cis-regulatory element Helix-loop-helix Zinc finger domain Leucine zipper domain

Extreme trans-acting effectors of transcription: TAL effectors From plant pathogenic bacteria Xanthomonas Secreted by bacteria when they infect Transcriptional activator-like (TAL) effectors bind with plant promoters to express genes beneficial for the bacteria

Repression of transcription TFs that act as repressors

Some trans-acting elements prevent transcription

Introducing RNAi H5udFjWDM3E&feature=related

What is a microRNA (miRNA)? Controlling gene expression post-transcriptionally. microRNA is an abundant class of newly identified small non-coding regulatory RNAs. Major characteristics of miRNAs: nt in length with a majority of nt non-coding RNA derived from a precursor with a long nt sequence this precursor can form a stem-loop 2 nd hairpin structure the hairpin structure has low minimal free folding energy (MFE) and high MFE index Slide courtesy of Baohong Zhang, East Carolina Univ

miRNA regulates plant development WT miRNA miRNA 156 increasing leaf initation, decreasing apical dominance, and forming bushier plant. miRNA 164 stamens are fused together. miRNA 172 sepal and petal disappeared. miRNA 319 Leaf morphology Slide courtesy of Baohong Zhang, East Carolina Univ

Small interfering RNAs inhibit expression of a homologous gene

Biogenesis of miRNAs Bartel, Cell. PlantAnimal

Post-transcriptional gene regulation Two major molecular mechanisms Mechanisms of miRNA-mediated gene regulation Zhang et al Developmental Biology Slide courtesy of Baohong Zhang, East Carolina Univ

Mary-Dell Chilton Undergrad and PhD University of Illinois Postdoc with Gene Nester and Milt Gorgon Univ Washington One of the first transformed plants Washington University Career at Ciba  Novartis  Syngenta

Pre-transcriptional gene regulation by methylation of DNA and acetylation of histones

Special proteins (e.g. chromomethylases) maintain methylation patterns

Switching a gene on and off through DNA methylation and histone modification

Arabidopsis MET1 Cytosine Methyltransferase Mutants Kankel et al (3):1109 Genetics Plants mutant for MET1 show late-flowering phenotypes

Histone acetyl transferases and chromatin remodeling allows promoters to be accessible to RNAPII

Figure 6.9

Some post-translational modifications Phosphorylation Biotinylation Glycosylation Acetylation Alkylation Methylation Glutamylation Glycylation Isoprenylation Lipoylation Phosphopantetheinyl ation Sulfation Selenation C-terminal amidation

Protein glycosylation in the ER

The central dogma revisited The order of the DNA template or coding strand is 3’ to 5’ This determines the order of the mRNA strand (5’ to 3’) because DNA template is complementary to the mRNA strand.

Figure 6.5 Eukaryotic gene structure and transcription of DNA into mRNA TF AAAAA RNA polymerase II TF

Manipulating gene expression Can be done at several levels –Promoters, enhancers, transcription factors –Post-transcriptional –Translational –Methylation Biotechnology typically manipulates promoter Post-transcriptional gene silencing (RNAi) increasingly important