molecular genetics & gene expression mat halter and neal stewart 2016
TRANSCRIPT
Molecular genetics & gene expression
Mat Halter and Neal Stewart2016
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 Crick James WatsonFigure 6.2http://www.stern.de/_content/50/44/504453/watson_crick_500.jpg
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?
DNA RNA Protein
Transcription
The Central Dogma
Figure 6.5
Eukaryotic gene structure
Transcription of DNA into mRNA
TF
TFAAAAA
RNA polymerase II
TFTF
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
http://www.forschenistkunst.at/bildbewertung/objekt35.php?id=35
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
Promoter LacZ LacY LacA
mRNA
VIDEO
Transcription of DNA into mRNA
TF
TFAAAAA
RNA polymerase II
TFTF
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?
https://www.youtube.com/watch?v=D3fOXt4MrOM
Transcription and translation, the movie.
Translation
DNA RNA Protein
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)AminoAcid
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 Acids Three-Letter Abbreviation One-Letter AbbreviationAlanine Ala AArginine Arg RAsparagine Asn NAspartate Asp DCysteine Cys CGlutamine Gln QGlutamate Glu EGlycine Gly GHistidine His HIsoleucine Ile ILeucine Leu LLysine Lys KMethionine Met MPhenylalanine Phe FProline Pro PSerine Ser SThreonine Thr TTryptophan Try WTyrosine Tyr YValine Val V
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-helixZinc 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
http://www.sciencemag.org/content/333/6051/1843/F2.large.jpg
Repression of transcription
TFs that act as repressors
Some trans-acting elements prevent transcription
Introducing RNAi
http://www.youtube.com/watch?v=H5udFjWDM3E&feature=related
What is a microRNA (miRNA)?What is a microRNA (miRNA)?Controlling gene expression post-transcriptionally.Controlling gene expression post-transcriptionally.
microRNA is an abundant class of newly identified small microRNA is an abundant class of newly identified small non-coding regulatory RNAs.non-coding regulatory RNAs.
Major characteristics of miRNAs:
• 18-26 nt in length with a majority of 21-23 nt
• non-coding RNA
• derived from a precursor with a long nt sequence
• this precursor can form a stem-loop 2nd 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 miRNAsBiogenesis of miRNAs
Bartel, 2004. Cell.
Plant Animal
Post-transcriptional gene regulation
Two major molecular mechanisms
Mechanisms of miRNA-mediated gene regulationMechanisms of miRNA-mediated gene regulation
Zhang et al. 2006. Developmental BiologySlide 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 CibaNovartisSyngenta
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. 2003. 163 (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
TFAAAAA
RNA polymerase II
TFTF
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