BIO 121 – Molecular Cell Biology Lecture Section 2 A. Regulation of Transcription B. Regulation of RNA and Protein Processing C. Protein Structure and Function

A. Cell Specialization: Regulation of Transcription Cell specialization in multicellular organisms results from differential gene expression

A. CELL SPECIALIZATION: Regulation of Transcription 1.Chromosome, Gene and RNA Architecture 2.Cell-Specific Regulation of Chromosome Structure 3.Cell-Specific Regulation of Transcription Activation

1. Review of Chromosome, Gene and RNA Architecture a. Review of Chromatin Structure b. Chromosomal Gene Arrangement c. Single Gene Components d. Nuclear RNA, mRNA and Protein e. Other RNA Molecules f. Fast review of Transcription

a. Review of Chromatin Structure Chromatin is a complex of DNA and protein in the eukaryotic nucleus Loosely packed chromatin is called euchromatin Dense packing of the heterochromatin makes it difficult for the cell to express genetic information coded in these regions Histones are proteins that are responsible for the first level of DNA packing in chromatin

DNA double helix (2 nm in diameter) Nucleosome (10 nm in diameter) Histones Histone tail H1 DNA, the double helixHistones Nucleosomes, or “beads on a string” (10-nm fiber)

Figure 4-65 Molecular Biology of the Cell (© Garland Science 2008)

Figure 4-11 Molecular Biology of the Cell (© Garland Science 2008) b. Chromosomal Gene Arrangement Humans: 23 chromosome pairs 3 billion bases ~24,000 genes

Figure 4-15 Molecular Biology of the Cell (© Garland Science 2008)

Figure 6-14 Molecular Biology of the Cell (© Garland Science 2008) Genes can reside on either strand

c. Single Gene Components Anatomy of a gene Exon means sequence that exits the nucleus Intron means sequence that stays inside the nucleus

d. Nuclear RNA, mRNA and Protein

Figure 6-21 Molecular Biology of the Cell (© Garland Science 2008)

e. Other RNA Molecules 1. The Translational Apparatus 2. Nuclear Effectors 3. Cytosolic Effectors

Table 6-1 Molecular Biology of the Cell (© Garland Science 2008)

DNA molecule Gene 1 Gene 2 Gene 3 DNA template strand TRANSCRIPTION TRANSLATION mRNA Protein Codon Amino acid Transcription: “To transcribe” to copy in the same language Translation: “To translate” to copy into a new language Templated Polymerization f. Fast review of transcription

Figure 6-8a Molecular Biology of the Cell (© Garland Science 2008) RNA Polymerase II Complex Does it All 12 Protein Subunits in Human

As RNA polymerase moves along the DNA, it untwists the double helix, 10 to 20 bases at a time Transcription progresses at a rate of 40 nucleotides per second in eukaryotes The large subunit of RNA Pol II caps and polyadenylates the nascent nRNA The same large subunit of RNA also links to the splicosome to facilitate subsequent processing Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Figure 6-9 Molecular Biology of the Cell (© Garland Science 2008) Multiple RNA Pol II molecules can read DNA simultaneously

So, how do individual cells regulate which of the genes in their genome they will express? Remember from Intro Bio that prokaryotes regulate expression through repressors/activators Eukaryotes have more complex regulatory mechanisms Histone modification regulates chromatin structure DNA modification regulates promoter accessibility Epigenetic modification can be copied and inherited Transcription factors regulate promoter activation Specialized transcriptional activities

2. Nucleosome and Histone Modification Regulates of Chromatin Structure Chromatin is a complex of DNA and protein in the eukaryotic nucleus Loosely packed chromatin is called euchromatin Dense packing of the heterochromatin makes it difficult for the cell to express genetic information coded in these regions Histones are proteins that are responsible for the first level of DNA packing in chromatin

Cell-specific control of chromosome structure Eukaryotic cells can systematically control which genes are available for expression. Our DNA is complexed 50:50 with proteins and is very highly regulated by enzymatic alterations of what is open and closed.

Figure 4-28 Molecular Biology of the Cell (© Garland Science 2008) Spontaneous nucleosome unwrapping

Figure 4-29 Molecular Biology of the Cell (© Garland Science 2008) ATP-dependent nucleosome unwrapping

Histones are covalently modified to control gene accessibility The methylation and/or acetylation of either histones or the DNA itself determines what promoters are exposed. Different cell types have different enzymes and, thus, different areas of protein and DNA are targeted for alteration

Cell-specific control of chromosome structure 3 Stages of Transcription 1. Initiation 2. Elongation 3. Termination Acetylation promotes InitiationMethylation can go either way (lysine amino acid residues)

3. The “Histone Code” Hypothesis Combinations of covalent modifications have specific information for the cell – This DNA is newly replicated – This DNA is damaged and needs repair – Express this DNA – Put this DNA into heterochromatin storage

Figure 4-43 Molecular Biology of the Cell (© Garland Science 2008)

Figure 4-45 Molecular Biology of the Cell (© Garland Science 2008)

Figure 4-46a Molecular Biology of the Cell (© Garland Science 2008)

b. Direct covalent modifications of DNA can also control expression from genes in the euchromatin

Methylation of globin genes in human embryonic blood cells

c. Heritability: Epigenetic Memory OK. So a cell differentiates to become a blood vessel smooth muscle cell or fibroblast How come all of its mitotic descendents don’t have to go through differentiation? – Trithorax proteins bind to open nucleosomes and keep them open. – Polycomb proteins methylate uneeded nucleosomes and then bind to them to keep them tight. – These effects can then be directly passed through mitotic cell division to the offspring.

Two DNA methyltransferases are important in modifying DNA

d. Transcription factors regulate promoter activation – Core promoter made up of TATAbox and CpG islands Site of RNA Pol II recruitment and activation TF II family transcription factors bind RNA Pol II to core – Tissue-specific TF are true transcriptional determinant for the cell type Bind to core promoter elements and distal enhancers Create binding sites for TF II family TF and stabilize Transcription Initiation Complex TS-TF also recruit histone acetyltransferases to expose DNA

Tissue-Specific Transcription Factor Families

Fig Promoter TATA box Start point Template DNA strand General Transcription factors RNA polymerase II Cell-Specific Transcription factors RNA transcript Transcription initiation complex The Transcription Initiation Complex forms on every gene that gets expressed. Its presence there is really determined by the tissue specific transcription factors that bind to enhancer cis-elements.

RNA polymerase is stabilized on the promoter site of the DNA by transcription factors recruited by promoters and enhancers

Fig. 17-7b Elongation RNA polymerase Nontemplate strand of DNA RNA nucleotides 3 end Direction of transcription (“downstream”) Template strand of DNA Newly made RNA 3’ 5’ 5

Figure 6-3 Molecular Biology of the Cell (© Garland Science 2008) The stability of the initiation complex determines how many transcripts

Tissue-specific transcription factors may bind different enhancers The pax-6 gene has four enhancers and is expressed exclusively in those four tissue types.

TS TFs can even control differentiation stability A really important idea in cell differentiation is that there must be a molecular mechanism that keeps a cell differentiated. – The pax-6 gene has a Pax-6 site in its enhancer – When it is present the transcription rate is maximal – This mechanism is repeated in several cell types – A rare positive feedback loop

e. Specialized transcriptional activities Only about 3-5% of RNA in a cell is mRNA Up to 80% of RNA is ribosomal RNA – As many as 10 million ribosomes per cell – humans have 400 rRNA gene copies on 5 chromosome pairs (frogs have 1200) – 4 eukaryotic subunits: 18S, 5.8S, 28S, 5S – First 3 from one gene with RNA Pol I – 5S is from a separate gene with RNA Pol III

Figure 6-42 Molecular Biology of the Cell (© Garland Science 2008)

Table 6-1 Molecular Biology of the Cell (© Garland Science 2008)

RNA Molecules and their RNA Polymerases Most snRNA and miRNA: Pol II tRNA, shRNA, snRNA 6, miRNA: Pol III snoRNA often encoded in introns: Pol II

B. CELL SPECIALIZATION: RNA and Protein Regulation 1.nRNA to protein (review) 2.Cell-Specific Regulation of mRNA Production 3.Cell-Specific Regulation of Peptide and Protein Production

1. nRNA to protein (review) nucleus cytosol

Fig Second mRNA base First mRNA base (5 end of codon) Third mRNA base (3 end of codon) 20 amino acids 64 codons: 61 = code for amino acids 3 = stop signals Genetic code is redundant (degenerate base) No codon specifies >1 unique amino acid Genetic code is nearly universal (a few exceptions) Must be read in frame (like words in a book) The Genetic Code

Fig Polypeptide Ribosome Amino acids tRNA with amino acid attached tRNA Anticodon Trp Phe Gly Codons 3 5 mRNA Key Players in: Translation - mRNA - tRNA - ribosome - amino acids

Translation determines the primary structure Primary structure determines the repetitive folding of the secondary structure Tertiary structure arises due to complex folding Quaternary structure arises due to the joining of multiple peptide chains subunits The latter two are the result of post-translational changes to the primary sequence

Fig. 5-21a Amino acid subunits + H 3 N Amino end Primary Structure Primary structure, the sequence of amino acids in a protein, is like the order of letters in a long word Primary structure is determined by inherited genetic information

a. Basic protein structure comes from the amino acid conformation that gives the lowest free energy most of this is driven by the polar aqueous and non-polar membrane phases

Figure 3-5 Molecular Biology of the Cell (© Garland Science 2008) In the aqueous phase polar side chains face out, in the membrane they are hidden

Fig. 5-21c Secondar Structure  pleated sheet  helix The coils and folds of secondary structure result from hydrogen bonds between repeating constituents of the polypeptide backbone

Fig. 5-21f Polypeptide backbone Hydrophobic interactions and van der Waals interactions Disulfide bridge Ionic bond Hydrogen bond Tertiary structure is determined by interactions between R groups, rather than interactions between backbone constituents Strong covalent bonds called disulfide bridges may reinforce the protein’s structure

Fig. 5-21g 3 polypeptides  Chains  Chains Collagen Hemoglobin Quaternary structure results when two or more polypeptide chains form one macromolecule - It is hard to predict a protein’s structure from its primary structure - Most proteins go through several states on the way to stable structure

2. Cell-Specific Regulation of mRNA Production a. Co/post-transcriptional RNA modification can effect amount and type of protein expressed 1. 5’ Capping and 3’ Polyadenylation determine how the nRNA will be handled 2. Splicing different mRNAs from the same nRNA using different exons allows cells to choose the protein they will make

Figure 6-22a Molecular Biology of the Cell (© Garland Science 2008) Formation of the 5’ Cap in mRNA

The roles of the 5’ Cap Allows the cell to distinguish mRNA from other RNA Allows for processing and export of the mRNA Allows for translation of the mRNA in the cytosol

Figure 6-37 Molecular Biology of the Cell (© Garland Science 2008) Formation of the 3’ PolyA tail in mRNA The position of the tail is coded in DNA

Many proteins have alternative poly-A sites which can either change the regulation of expression at the 3’UTR or, less commonly, change the length of the coding region. The choice of poly-A site can be regulated by external signals

The roles of the 3’ Poly-A Tail Regulates termination of transcription Regulates nuclear transport Regulates the initiation of translation Controls the total amount of translation

2. Splicing different mRNAs from the same nRNA using different exons allows cells to choose the protein they will make – Alternative splicing occurs in ~92% of human genes – “Splice sites” are formed from consensus sequences found at the 5’ and 3’ ends of introns – Different splicosome proteins made in different cells recognize different consensus sequences – The result is families of related proteins from the same gene in different cell types

Fig Pre-mRNA mRNA Coding segment Introns cut out and exons spliced together 5’ Cap Exon Intron 5’ ExonIntron 105 Exon 146 3’ Poly-A tail 5’ Cap 5’ UTR3’ UTR RNA splicing removes introns and joins exons, creating an mRNA molecule with a continuous coding sequence

Examples of alternative RNA splicing

Alternative RNA splicing to form a family of rat α- tropomyosin proteins

The Dscam gene of Drosophila can produce 38,016 different types of proteins by alternative splicing The proteome in most eukaryotes dwarfs the genome in complexity!

Dscam protein is required to keep dendrites from the same neuron from adhering to each other Dscam complexity is essential to the establishment of the neural net by excluding self-synapses from forming

Differential RNA Processing Splicing Enhancers and Recognition Factors - These work much like transcription enhancers and factors - Enhancers are RNA sequences that bind factors to promote or silence spliceosome activity at splice site - Many of these sequences are cell type-specific, eg. muscle cells have specific sequences around all of their splice sites, thus make muscle- specific variants - Trans-acting proteins recognize these sequences and recruit or block spliceosome formation at the site

Muscle hypertrophy through mis-spliced myostatin mRNA Splice site mutations can be very deleterious, rarely can be advantageous

Fig RNA transcript (pre-mRNA) Exon 1Exon 2Intron Protein snRNA snRNPs Other proteins 5 5 Spliceosome components Cut-out intron mRNA Exon 1 Exon 2 5

Differential RNA Processing Spliceosome proteins link directly to the nuclear pore to facilitate transfer of the spliced mRNA into the cytosol

Proteins often have a modular architecture consisting of discrete regions called domains In many cases, different exons code for the different domains in a protein Exon shuffling may result in the evolution of new proteins Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Alternative splicing can have very powerful effects on protein function

Fig Gene DNA Exon 1Exon 2 Exon 3 Intron Transcription RNA processing Translation Domain 2 Domain 3 Domain 1 Polypeptide

Figure 3-12 Molecular Biology of the Cell (© Garland Science 2008) Because of gene duplication and exon duplication modularity of structure is common: Protein families and domains Two serine protease genes give nearly exact binding site structure

b. Selective Degradation of RNA 1.Prevention of export of incomplete or intronic RNA from the nucleus 2.Prevention of translation of damaged or unwanted RNA in the cytosol

Cell type 1Cell type 2 2. Cytosolic selection

1. Prevention of export of incomplete or intronic RNA from the nucleus – More genes are transcribed in the nucleus than than are allowed to be mRNA in the cytosol – The unused nRNAs are degraded in the nucleus or used to make non-coding RNA molecules

At every step in the processing of the transcript it must lose and/or gain the appropriate proteins to be identified as ‘ready’. Positive for export:cap and PolyA binding proteins exon junction complex SR proteins, nuclear export receptor Negative for export: snRNP Positive for translation: translation initiation factors Negative for translation: cap binding protein The inappropriate combination of markers leads to degradation by nuclear exosome and cytosolic exonuclease

Figure 6-80 Molecular Biology of the Cell (© Garland Science 2008) Eukaryotic block to translation: Unbalanced EJC’s

2. Prevention of translation of damaged or unwanted RNA in the cytosol a.Failed recognition of 5’-cap and poly-A tail prevents translation-initiation machinery b.Recognition of defective mRNA c.Bacteria have quality control mechanisms to deal with incompletely synthesized and broken mRNAs

Figure 6-81 Molecular Biology of the Cell (© Garland Science 2008) Prokaryotic block to translation

3. Cell-Specific Regulation of Peptide and Protein Production a.Regulation of translation b.Co-/Post-translational protein regulation

a. Regulation of translation 1.5’ and 3’ untranslated regions of mRNAs control their translation 2.Global regulation of translation by initiation factor phosphorylation 3.Small noncoding RNA transcripts regulate many animal and plant genes 4.RNA interference is a cell defense mechanism

1. 5’ and 3’ untranslated regions of mRNAs control their translation a.Translation initiation is controlled at the 5’-cap b.Alternative sites of translation initiation are controlled at internal ribosome entry sites c.The amount of protein translated from mRNA is controlled by mRNA stability 1.Cytoplasmic poly-A addition can regulate translation 2.External factors can extend RNA life

a. A normal 5’-cap allows assemblage of the translation initiation complex small ribosomal subunit

b. Internal ribosome entry sites provide alternative sites of translation initiation Multiple AUG start codons in one mRNA sequence A given cell can choose one or the other by it the translation initiation factors it expresses

Figure Molecular Biology of the Cell (© Garland Science 2008) normalalternative

Figure 6-3 Molecular Biology of the Cell (© Garland Science 2008) c. 5’ caps and 3’ poly-A tails dictate the duration of time that the mRNA is active in the cytosol

Figure Molecular Biology of the Cell (© Garland Science 2008) The length of the poly-A tail determines how long the mRNA survives Once the tail is degraded: Coding sequence is destroyed and/or The 5’ cap is removed

2. External factors can extend RNA life The length of translation can also respond to external regulation from hormones, growth factors, etc.

Degradation of casein mRNA in the presence and absence of prolactin

b. Co-/Post-translational protein regulation 1.Folding and membrane insertion 2.Covalent modifications 3.Polymer assembly 4.Proteolytic modifications

1. Folding and membrane insertion Molecular chaperones help guide the folding of most polypeptides while still being synthesized – Heat shock proteins (Hsp) Hsp70 (BIP) Hsp60 (chaperonins) – Calnexin, calreticulin – “Folding”, “Protease Inhibitor”

Figure 6-86 Molecular Biology of the Cell (© Garland Science 2008)

Fig Hollow cylinder Cap Chaperonin (fully assembled) Polypeptide Steps of Chaperonin Action: An unfolded poly- peptide enters the cylinder from one end The cap attaches, causing the cylinder to change shape in such a way that it creates a hydrophilic environment for the folding of the polypeptide. The cap comes off, and the properly folded protein is released. Correctly folded protein

Figure 12-43c Molecular Biology of the Cell (© Garland Science 2008) Many membrane proteins are associated with the lipid bilayer during translation

Figure (part 2 of 2) Molecular Biology of the Cell (© Garland Science 2008)

Figure Q12-5 Molecular Biology of the Cell (© Garland Science 2008)

Figure 6-90 Molecular Biology of the Cell (© Garland Science 2008) Misfolded proteins are controlled by regulated destruction proteasome

Figure Molecular Biology of the Cell (© Garland Science 2008)

2. Covalent Modifications Glycosylation by cell-specific enzymes can change the function of a shared protein Different kinases in different cells may phosphorylate proteins at alternative sites Isomerization of disulfide linkages in different cells can produce different functions Variability in methylase/acetylase proteins can dramatically alter cell phenotype and function

Figure 19-60b Molecular Biology of the Cell (© Garland Science 2008)

Figure 3-27a Molecular Biology of the Cell (© Garland Science 2008) 3. Polymer Assembly

Figure Molecular Biology of the Cell (© Garland Science 2008) 42 genes in humans for  -collagen You need three to make a protein 40 different proteins have been shown

Figure 3-35 Molecular Biology of the Cell (© Garland Science 2008) 4. Proteolytic Modifications

C. Protein Structure and Function A protein’s biochemistry dictates its functional activities Regulation of protein structure and function is one of the most fundamental means by which cells control their own activities

Protein Structure = Function Functional amino acids often widely distributed The cell can control protein activity by regulating amino acid position

EXTRACELLULAR FLUID [Na + ] high [K + ] low Na + [Na + ] low [K + ] high CYTOPLASM Cytoplasmic Na + binds 1 Example: The sodium- potassium pump Binding changes protein conformation, change in conformation alters activity

Na + binding stimulates phosphorylation by ATP. Fig Na + ATP P ADP 2

Fig Phosphorylation causes the protein to change its shape. Na + is expelled to the outside. Na + P 3

Fig K + binds on the extracellular side and triggers release of the phosphate group. P P K+K+ K+K+ 4

Fig Loss of the phosphate restores the protein’s original shape. K+K+ K+K+ 5

Fig K + is released, and the cycle repeats. K+K+ K+K+ 6 Why do we want Na + outside of the cell and K + inside?

2. Cells can control a protein’s activity directly - by mechanisms that target the protein itself

Figure 3-58 Molecular Biology of the Cell (© Garland Science 2008) a. Definition: “Allosteric”. Proteins with two or more binding sites, wherein activity away from the active site will regulate activity at the active site

Figure 3-59 Molecular Biology of the Cell (© Garland Science 2008)

Figure 3-64 Molecular Biology of the Cell (© Garland Science 2008) b. Cells can start and stop a protein’s activity by changing its structure through the addition of a covalent subgroup phosphorylation-dephosphorylation

Figure Molecular Biology of the Cell (© Garland Science 2008) glycosylation

Figure Molecular Biology of the Cell (© Garland Science 2008) Addition of covalently linked lipids allows a protein to have a tight association with the membrane

Complex Covalent Regulation of the p53 Transcription Factor

Figure 3-35 Molecular Biology of the Cell (© Garland Science 2008) c. Cells can start and stop a protein’s activity by proteolytic cleavage

Figure 18-5a Molecular Biology of the Cell (© Garland Science 2008) The clotting cascade is the same

3. Cells can control protein activity indirectly by altering the other molecules that share its environment

a. Cells can start and stop a protein’s activity by regulating the presence of a critical binding partner 1. Ligand interaction activates receptor

Figure Molecular Biology of the Cell (© Garland Science 2008) 2. Sequestration of effector molecules linked to controlled release eg. Hide all of the calcium until you want to change actin-myosin activity

3. Cooperative/Coupled binding - Substrate binding in one site effects binding of substrate in second site by changing affinity Hemoglobin binds oxygen with greater affinity when there is lots of oxygen – this ensures flow of oxygen to the tissues and not away.

Figure 16-78a Molecular Biology of the Cell (© Garland Science 2008) b. Cells can start and stop a protein’s activity by blocking its binding site Tropomyosin blocking the myosin-binding site on actin

Figure 17-49a Molecular Biology of the Cell (© Garland Science 2008) c. Cells can start and stop a protein’s activity by regulating the processes that make active polymers from inactive subunits

Figure 3-79 Molecular Biology of the Cell (© Garland Science 2008) d. Cells can start and stop a protein’s activity by regulating the scaffolded interaction of the subunits of protein machines Ubiquitin ligase complex

Figure Molecular Biology of the Cell (© Garland Science 2008) Synaptic scaffold

Figure 15-21a Molecular Biology of the Cell (© Garland Science 2008)

Figure Molecular Biology of the Cell (© Garland Science 2008) Sometimes the scaffold protein is even part of the signaling cascade!