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Stem cells characteristics
- 1. Self-Renewal-ability to divide to produce more stem cells
- 2. Pluripotent-to differentiate into specialized cell types.
Multipotent stem cells
give rise to all cell types in a tissue
Hydrophilic amino acids
- (acidic, basic and uncharged)
- generally found at the surface of water-soluble proteins or protein domains
Hydrophobic amino acids
- (linear, branched and aromatic)
- generally found in theinterior of water-soluble proteins or in lipid-associated regions of proteins
- sulfhydryl group (SH) of a cysteine can form a covalent disulfide bond with the SH group of another cysteine
- Disulfide bonds can occur within a protein or between proteins.
- the R group is a hydrogen, making glycine the smallest amino acid
- Glycine therefore causes little steric hindrance and allows structural flexibility.
- amino group is covalently joined to the side chain, forming a ring structure that makes proline rigid.
- interrupts and α-helix
- Proteins fold into the thermodynamically most stable conformation
- determined by interactions between amino acid residues
- regular coil structure stabilized by hydrogen bonds between the peptide bond carbonyl group and the peptide bond amide four residues towards the carboxy-terminus.
- The R groups project outwards.
- Proline interrupts an α-helix.
- lateral (side-by-side) association of β strands.
- β strands are linear, extended stretches of amino acids.
- Strands are joined by hydrogen bonds between carbonyl groups on one strand and amide groups on other.
- Strands can be parallel or anti-parallel. R groups project up or down.
- 3-4 aa U-shaped turn stabilized by hydrogen bonds between carbonyl group of first residue and amide group of last residue.
Supersecondary loop stabilized by binding to Zn+2 ion (in some transcription factors, other proteins).
- supersecondary two α-helices wound around one another.
- Hydrophobic side chains on every 1st and 4th aa interdigitate.
- Often mediate protein-protein interactions.
- cooperative binding and dissociation of oxygen which makes hemoglobin more suited for oxygen delivery to tissues than myoblobin.
- This type of effect on substrate (oxygen) binding by an interaction at another site (binding to another hemoglobin chain) is known as an allosteric effect.
Integral membrane proteins
- embedded (pass through) the lipid bilayer
- transmembrane proteins
- region that spans the membrane is usually an α helix composed of hydrophobic amino acids.
- The protein regions on either side of the membrane use the same organizing principals as soluble proteins. Integral membrane proteins are often glycosylated on the lumenal (non-cytoplasmic) domains.
- Sugar chains are covalently linked to the NH of asparagine (“N-linked”) or the OH groups of serine and/or threonine (“O-linked”).
- Some integral membrane proteins do not have hydrophobic transmembrane domains but instead have covalently attached lipids that insert into the bilayer and act as anchors.
Peripheral membrane proteins
- associated with the surface of membranes but do not extend into the hydrophobic core of the bilayer.
- Association usually involves interaction with a transmembrane protein and/or with the hydrophilic head groups of the membrane lipids.
Genetics of disease
- MULTIFACTORIAL-some disease and cancer are due to the combined effects of mutations in multiple genes (polygenic), often combined with environmental factors
- SOMATIC-in cells other than the germ-line then it is not passed on to offspring but it is passed on to progeny of the mutant cell in the individual as the cell divides
- GERM-LINE-disease can be inherited
- different forms of a gene.
- The normal allele of a gene is often referred to as the wild-type allele.
Human males have only one copy of alleles on the Y chromosome and this is referred to as hemizygous
same allele on both chromosomes
- both alleles have to be mutant in order to see a mutant phenotype
- Recessive mutations normally cause inactivation or elimination of a gene/protein.
- That is, they cause a loss of function.
- Ex cystic fibrosis
- one normal copy of a gene does not produce enough protein to prevent disease
- dominant mutation
- If the mutant allele produces a form of the protein that INTERFERES with the function of the normal protein, often by binding to the normal protein, then this could cause disease in a heterozygote.
- If the mutant allele produces a protein with NEW, or INCREASED levels, of function , then this could cause disease in a heterozygote.
- This type of mutation is referred to as dominant-positive or gain-of function.
Mitochondria are inherited from the egg so mitochondrial disorders are inherited only from the mother (trace maternal lineage)
- Prions are infectious agents, consisting only of protein, that have the ability to reproduce within cells
- exception to the dogma that infectious agents (like viruses, bacteria, etc) require nucleic acids for reproduction.
- Prions cause bovine spongiform encephalopathy (BSE/mad cow disease) and certain slow, nervous system degeneration diseases in humans (Creutzfeldt-Jakob disease, fatal familial insomnia).
- Prions are abnormally folded forms of endogenous protein that can convert the endogenous form into the abnormal form.
- The abnormal form is in a mostly β-sheet conformation while the normal form is mostly α-helical.
- The normal and abnormal forms have exactly the same amino acid sequence, but different secondary and tertiary structures.
- an organic base (adenine, guanine, thymine, cytosine, or uracil) is linked by an N-glycosidic bond to the 1’ carbon atom in a 5 carbon sugar.
- In DNA the sugar is 2’ deoxyribose while in RNA the sugar is ribose.
- The sugar contains a phosphate group in ester linkage with the 5’ carbon
- (adenine and guanine, abbreviated A and G
- 2 rings
have 1,2, or 3 phosphates esterified to the 5’ carbon.
do not have any 5’ phosphate. Base & Sugar
5' to 3'
- chain of DNA has a polarity –
- 5’ end has a free phosphate or hydroxyl at the sugar’s 5’ carbon
- 3’ end has a free hydroxyl on the sugar’s 3’ carbon.
- A pairs with T through two H bonds
- G to C through 3 H bonds
- The specificity of base-pairing is referred to as complementarity
- The major form of DNA in the cell is known as the B form.
- the A form is found in RNA/DNA helices and is more compact
- A and B DNA forms righthanded helices.
- Z form is left-handed and the backbone is “zig-zagged” and can form in regions where there are alternating Gs and Cs
Strand separation (often called denaturation or melting) occurs during DNA replication and transcription.
Re-pairing of the complementary strands is known as renaturation or annealing.
Denaturation and renaturation are the basis of an important molecular biology technique known as hybridization. Hybridization is used to detect specific nucleotide sequences in a mixture of DNA with different sequences.
DNA is specially packaged into a compact DNA and protein complex called chromatin
- basic structural unit of chromatin is the nucleosome
- consists of DNA wrapped around a protein core of histones, which are small basic proteins.
- Each core is octameric and contains 2 copies each of histones H2A, H2B, H3 and H4.
- About 145 bp of DNA is wrapped twice around the protein core.
- In a chromosome the nucleosomes are arranged as “beads on a string” with 15-55 bp of DNA linking each one nucleosome to the next.
- Histone H1 binds to the linker region and helps pack the nucleosomes into a higher order solenoid.
- The solenoid itself is arranged into loops along a central protein scaffold
- 1) as loops on the scaffold (euchromatin, transcriptionally active)
- 2) as more tightly packed DNA (heterochromatin, transcriptionally inactive).
- In heterochromatin the scaffold is folded into a helix which itself is further compressed into an undefined structure
- (untranslated regions – UTR):
- present at the ends of mRNA transcripts but do not encode amino acids.
- upstream of the initiator codon (5’ UTR)
- downstream of the termination codon (3’UTR).
- They play roles in controlling processing of the 3’ end, controlling translation, and controlling RNA localization in the cell.
- (intervening sequences)
- transcribed into RNA but are removed during formation of the mature mRNA (SPLICING).
- They can interrupt the coding region as well as 5’ and 3’ non-coding regions.
- Removal of introns is called splicing.
- The regions in the DNA and initial transcript that ultimately are retained in the mRNA are called exons
- Synthesis of RNA
- uses a DNA template and synthesizes a new strand in the 5’ to 3’ direction, copying the template in the 3’ to 5’ direction.
- The substrates are nucleoside triphosphates (NTPs).
- Pol I synthesizes rRNA.
- Pol II synthesizes mRNA and some small structural RNAs
- Pol III synthesizes tRNA and 5S rRNA.
- specific sequences in the 5’ ends of genes that serve as signals to position RNA polymerase at the proper site to begin transcription
- Since these sequences are located on the same DNA as the gene they are referred to as cis-acting elements.
- sequences are located on the same DNA as the gene
- promoter, TATA box
- In many eukaryotic genes a key cis-acting element is the sequence TATAAA located 18-34bp before the start site of transcription.
- The TATA box serves as a binding site for a general transcription factor that positions RNA polymerase at the 5’ end of the gene.
- Other sequences near the TATA box also bind other general transcription factors.
Promoter Proximal Elements
- other DNA sequences that are necessary for optimal transcription
- Some are located near to the TATA box.
- These are referred to as promoter proximal elements.
- In some cases these elements are cell-type specific. That is, they aid transcriptional initiation of a gene in some cells but not others.
- Other sequences can be located very far (thousands of base-pairs) from the promoter, either upstream or downstream of the gene. These are known as enhancers and they are frequently cell-type-specific.
- DNA sequence necessary for optimal transcription
- located very far (thousands of base-pairs) from the promoter, either upstream or downstream of the gene.
- These are known as enhancers and they are frequently cell-type-specific.
Both promoter-proximal elements and enhancers are bound by other transcription factors that increase the rate of transcriptional initiation. These factors are known as activators.
- Some DNA sequences are recognized by proteins that decrease the rate of transcription.
- The DNA elements are called silencers and the proteins are generally called repressors.
- General transcription factors are required at most promoters for transcription initiation by Pol II
- contains TATA-binding protein (TBP) which directly binds to the TATA box, plus associated proteins called TAFs
Transcription Factors Activators
- Pol II and TFs can carry out low (basal) levels of transcriptional initiation.
- Transcriptional activators bind to promoter-proximal and enhancer elements and increase the rate of transcriptional initiation.
- They act synergistically (more than additive) to increase transcription by increasing the binding of RNA pol II to the promoter.
- Activators generally consist of 2 domains:
- 1) a DNA binding domain that binds to the base-pairs in the promoter-proximal element or enhancer;
- 2) an activation domain that is responsible for transcriptional activation.
- Many activation domains do not interact directly with RNA PolII but instead interact with an intermediary complex (also known as a co-activator) called the Mediator complex.
- Mediator therefore bridges activators with RNA PolII.
- classified by their DNA binding domains.
- Most DNA binding domains contain an α helix that fits into the major groove of the DNA and makes specific hydrogen bonds with the base-pairs.
Activator that contains a conserved 60aa DNA binding domain that contains 3 helices. The transcription factors Msx1 and Msx2 that are mutated in familial tooth agenesis and craniosynostosis , type II are homeodomain proteins. In Boston type Craniosynostosis a mutation of proline to histidine in the homeodomain of Msx2 causes tighter DNA binding, possibly accounting for the dominant positive nature of the disease.
- bind silencer sequences in DNA
- lower transcription by preventing the binding of an activator to DNA or by preventing binding of activators to Mediator or by modifying chromatin structure.
Chomatin Structure Regulation
- Packing of chromatin into condensed structures can prevent transcription factors from gaining access to the DNA.
- enzymes that acetylate or deacetylate lysine residues in the N-terminal regions of histones
- Some activators function by binding histone acetylases.
- Some repressors function by binding histone deacetylases.
- Modifications of chromatin (and DNA) at specific genes can be stable through cell division.
- Chromatin (and DNA) modifications that are passed on to daughter cells are referred to as epigenetic because they alter the gene structure without changing the DNA sequence.
RNA Processing and Export
- Additional steps are required to produce a functional mRNA capable of being translated into protein.
- 1. 5’ cap addition.
- 2. transcription termination and polyadenylation
- 3. splicing to remove introns
- 4. transport from the nucleus to the cytoplasm.
5’ cap addition
- Soon after the transcript is initiated the 5’ end is modified by addition of a guanosine nucleotide in an unusual 5’ to 5’ triphosphate ester bond.
- The guanosine is methylated on the N7 position after addition and the 2’ OH positions of the first two bases of the original transcript can also be methylated.
- The cap protects the 5’ end from degradation by exonucleases and it is involved in mRNA binding to the ribosome.
Termination and polyadenylation
- mRNAs have “tails” of about 200 As.
- Cleavage and poly A addition generally require the sequence AAUAAA in the 3’ untranslated region of the RNA transcript.
- A multiprotein complex recognizes AAUAAA and cleaves the RNA 20-50 bases downstream.
- Then poly A polymerase addsabout 200 As in a non-templated reaction.
- Poly A tails are thought to protect the 3’ end from degradation by exonucleases.
- Most eukaryotic genes contain regions that are included in the mature mRNA (exons) and regions that are excised from the initial transcript (introns).
- Introns are removed from the initial RNA transcript by splicing.
- Splicing must be precise because if the junction is off by even one base then the reading frame will be altered, resulting in a mutation.
splice donor site
- 5' Splice Site
- At the 5’ exon/intron boundary is the sequence AG/GU
Splice Acceptor Site
- 3' Splice Site
- At the 3’ intron/exon boundary is the sequence AG/G
Near the 3’ end of the intron is a sequence containing mostly pyrimidine nucleotides (pyrimidine-rich) and a nearby critical adenine nucleotide (called the branch point).
- made up of small nuclear ribonucleoprotein particles (snRNPs) that each contain a snRNA and 6-10 proteins.
- Since the RNAs have many U nucleotides, the snRNPs are referred to as U1-U6.
- U1 binds the 5’ splice site GU.
- U2 and an associated factor binds the branchpoint A and the pyrimidine-rich sequence.
- Then U4,5,6 bind to form a looped structure putting the A near the 5’ exon/intron boundary.
- The A forms a 2’ to 5’ phosphodiester bond with the first G of the intron which results in a looped RNA called a lariat.
- Then the 3’ end of the first exon is joined to the 5’ end of the next exon.
looped RNA of intron during splicing
- joining different combinations of exons can produce different mRNAs encoding different proteins
- An example is the fibronectin gene.
- Fibroblasts produce an “isoform” of fibronectin that adheres to the cell surface and is important in cell attachment to the extracellular matrix.
- Hepatocytes produce an isoform of fibronectin that does not adhere well to the cell surface and consequently circulates in the serum where it plays a role in blood clotting.
- The two forms are produced from the same gene by including (fibroblasts) or excluding (hepatocytes) two exons that encode protein regions involved in binding to the cell surface.
- 60% of all human genes are alternatively spliced.
- 25,000 genes but about 85,000 different mRNAs, suggesting that alternative splicing plays a key role in generating protein diversity in humans