BMS100 ClinPhys Central Dogma.docx

BMS100: CLINICAL PHYSIOLOGY REVIEW NOTES CENTRAL DOGMA DNA and RNA Structure Two types of nucleic acids: DNA: deoxyribonucleic acid; forms inherited genetic material inside our cells – genes code for protein and determine physical traits Two types of nitrogenous bases: Purines – double ring bas...

BMS100: CLINICAL PHYSIOLOGY REVIEW NOTES CENTRAL DOGMA DNA and RNA Structure Two types of nucleic acids: DNA: deoxyribonucleic acid; forms inherited genetic material inside our cells – genes code for protein and determine physical traits Two types of nitrogenous bases: Purines – double ring base (A, G) Pyrimidines – single ring base (C, U, T) Double-helix structure; sugar-phosphate backbone A pairs with T; G pairs with C Chargaff’s rule: number of purines must always equal number of pyrimidines Forces stabilizing DNA double helix: H-bonds between complimentary base pairs Sugar phosphate backbone – phosphodiester bond; magnesium helps stabilize Base stacking – parallel to each other for hydrophobic effects RNA: ribonucleic acid; polymer made of nucleotides linked by phosphodiester bond Differs from DNA in three main ways: Ribose sugar vs. deoxyribose sugar Uracil base rather than thymine Single stranded (not double) DNA is transcribed into RNA to serve as a template for protein translation = mRNA DNA Condensation Nucleosomes: structural unit for packaging DNA Composed of: 147 base pairs wrapped around a histone core (octamer of H2A, H2B, H3 and H4) and a H1 linker protein Chromatin: complex of DNA + tightly bound protein Can be found as heterochromatin (densely packed) or dispersed euchromatin (dispersed) Chromosomes: how DNA is packaged when it is in its most condensed form Most human cells = 23 pairs of chromosomes; 46 total 1 copy of each chromosome come from each parent (diploid) – maternal and paternal chromosomes called homologous chromosomes Haploid chromosomes – only egg and sperm cells Autosomal chromosomes – chromosomes 1-22; form homologous pairs Sex chromosomes – determine biological sex; non-homologous (XX, XY) Genes: functional units of heredity that is a segment of DNA containing the instructions for making a particular protein; chromosomes carry genes Exon => coding sequence Intron => non-coding sequence of a gene; removed via splicing after transcription 98.5% of human genome does not encode protein; regulates gene expression Ex. promotor and enhancer regions, binding sites that organize chromatin structures, non-coding regulatory RNA, mobile genetic elements (transposons) Types of RNA mRNA: messenger RNA; DNA is transcribed into mRNA to serve as a template for protein translation pre-mRNA: initially; then undergoes processing into mature mRNA Non-coding mRNA: sequences of DNA that are transcribed into RNA that does not get translated into proteins; serve as enzymatic, structural, and regulatory components snRNA: small nuclear RNA; functions in the spliceosome Spliceosome: needed to remove introns from pre-mRNA Associated with protein subunits to form small nuclear ribonucleoproteins (snRNPs) which form core of the spliceosome rRNA: ribosomal RNA; needed for basic structure of ribosome complex Involved in catalysis of the peptide bond between amino acids tRNA: transfer RNA; needed in translation to carry correct amino acid to the growing polypeptide chain; folds into unique cloverleaf shape Two important regions for protein translation: Anticodon: 3 consecutive nucleotides that pairs with complementary codon in an mRNA molecule Amino acid binding site: short single-stranded region at 3’end of the tRNA molecule that binds amino acid corresponding to the anti-codon on the tRNA miRNA: micro-RNA; regulate gene expression via post-translational silencing; block or prevent translation of specific mRNAs and promote their degradation siRNA: small interfering RNA; reduce gene expression by direct degradation of specific mRNA lncRNA: long non-coding RNA; can increase or decrease transcription – involved in X-chromosome inactivation Wobble Hypothesis Redundancy of genetic code: More than 1 possible tRNA for many of the amino acids Some tRNA molecule can base-pair with more than one codon Some tRNA are built to only require accurate base-pairing of only first two positions of the codon and can tolerate mismatch (wobble) in the third position Transcription Transcription: process of synthesizing an RNA molecule from DNA template (gene) that will dictate the synthesis of a protein; occurs in nucleus of a cell Transcription unit: outlines the 3 general regions found in all genes: Promoter region: contains consensus sequence (ex. TATA box) Coding region: transcribed into mRNA Terminator region: specifies end of transcription Template strand: strand of DNA that is transcribed into RNA (also anti-sense strand) Non-template strand: complimentary partner to template strand (also sense strand) RNA polymerase: key enzyme for transcription; moves along DNA to unwind the helix just ahead of active site for polymerization and catalyzes new phosphodiester bonds on the newly-forming strand of RNA Works in 5’ 3’ direction A to U T to A G to C 4 Stages of Transcription Initiation RNA polymerase recognizes where to start Initiation factors help with this process Prokaryotes = sigma factor (only one) Eukaryotes = ex. TFII (many types) Regulation of initiation: Repressor proteins: can bind upstream sequences called silencers (negative regulatory elements) that inhibit gene expression Enhancers: transcriptional activator proteins that bind upstream sequence (positive regulatory elements) which increase the rate of transcription by attracting RNA polymerase II enzyme TFII recognizes and binds consensus sequence in promoter region – ex. TFIID binds TATA box Other transcription factors join RNA polymerase II joins Transcription initiation complex complete Elongation RNA polymerase begins transcribing moving downstream along DNA coding region– most transcription factors (TFII) are released Eukaryotes require: Elongation factors: help reduce likelihood of RNA polymerase dissociating before reaching the end Chromatin remodeling complexes: help RNA polymerase navigate the chromatin structure Histone chaperones: partially disassemble and reassemble nucleosomes as an RNA polymerase passes through As RNA polymerase moves along it generates supercoils – DNA topoisomerase removes this super-helical tension DNA topoisomerase: relieves the super-helical tension by breaking phosphodiester bonds; allows two sections of the DNA helix to rotate freely and relieve tension (bond re-forms once it leaves) Processing In eukaryotes, pre-mRNA transcript is process in 3 main ways: Splicing Spliceosomes: remove introns by splicing; require snRNA and proteins complexed into snRNPs Allows same gene to produce a variety of different proteins Capping the 5’ end 7-methyl guanosine cap – a modified guanine nucleotide is added to 5’ end of the pre-mRNA 5’ cap facilitates export of mRNA into nucleus and involved in translation Polyadenylation of 3’ end Once cleaved, ~200 adenine (A) nucleotides are added to mRNA Poly-A tails protect mRNA from degradation and facilitates export from nucleus Poly-A binding proteins bind the poly-A tail Processing and termination 3’ end of the mRNA molecule is specified by signals encoded in DNA Signals are transcribed into RNA and bind to proteins that facilitate cleave of mRNA from RNA polymerase = once cleaved, poly-A tail is added^ *In prokaryotes mRNA transcript produced is different in: No processing required – no 5’ cap, splicing or poly-A tail No export from nucleus – translation begins right away mRNA transcript is polycistronic – codes for more than one protein Translation Translation: mRNA is translated into protein Mature mRNA from transcription exported through nuclear pore complexes into cytosol for translation Decoded in sets of 3 nucleotides = codons Reading frame: way of dividing the sequence of nucleotides in a nucleic acid molecule into triplets 64 possible combinations but only 20 amino acids – redundancy of genetic code Needed for translation: mRNA transcript tRNA – cell makes variety of tRNAs each corresponding to one of 20 amino acids; enzyme aminoacyl-tRNA synthetase catalyzes attachment of correct amino acid to tRNA Ribosomes – where protein synthesis is performed; helps maintain correct reading frame and ensure accuracy of codon-anti-codon interaction Ribosome complex composed of ribosomal proteins and ribosomal RNA (rRNA) 2 subunits – small and large Polysomes: synthesis of proteins occurs on polyribosomes (polysomes) As soon as preceding ribosome has translated enough of the nucleotide sequence to move out of the way – a new ribosome complex is formed Helps speed up rate of protein synthesis 3 Stages of Translation Initiation AUG – first codon translated on mRNA Initiator tRNA carries amino acid methionine (all proteins begin with methionine at N-terminus) = forms met-tRNAi complex Met-tRNAi is loaded into small ribosomal subunit with initiation factors (eIFs) Small ribosome binds the 5’ end of the mRNA (cap helps with recognition) – moves along mRNA from 5’ to 3’ scanning for the first AUG; requires ATP hydrolysis Initiation factors dissociate and large ribosome subunit assembles to complete ribosome complex Elongation Elongations proceeds efficiently and accurately with help of elongation factors (EFs); enter and leave ribosome during each cycle and are coupled with GTP hydrolysis tRNA binding – newly charged tRNA binds to A site of the ribosome complex Peptide bond formation – carboxyl end of polypeptide chain released from tRNA at the P site and joins amino acid linked to the tRNA at the A site Peptide bond is catalyzed by peptidyl transferase enzyme contained within the large ribosomal subunit Translocation of large subunit – two tRNAs are shifted to the E and P sites Translocation of small subunit – small subunit shifts by 3 nucleotides; tRNA in E site is ejected Termination STOP codon marks end of translation – UAA, UAG, UGA Release factors bind to ribosomes with a stop codon in the A site Peptidyl transferase catalyzes the addition of water rather than amino acid – frees carboxyl end and releases polypeptide Ribosome releases the mRNA and dissociates into the large and small subunits (recycled) *In prokaryotes: Additional recognition sequence is needed for ribosome binding = Shine-Dalgarno sequence (ribosome-binding site) Post-translational Protein folded into specific 3D shape May be modified in the ER – ex. glycosylated Sent to proper cellular location Regulation of Protein Synthesis Cell can regulate the amount of a particular protein available to a cell by regulating: Amount of transcription that occurs Histones: regulated by nuclear proteins; can modify proteins Regulators of histones: Chromatin remodeling complexes – reposition nucleosomes on DNA to expose or obscure gene regulatory elements Chromatin writer complexes – carry out histone modifications; methylation, acetylation or phosphorylation Histone acetylation tends to open chromatin and increase transcription; performed by histone acetyltransferases; deacetylation reverses change and promotes chromatin condensation Histone methylation can promote transcriptional activation or repression; typically results in transcriptional silencing Stability of mRNA transcript The longer mRNA lasts in cytosol = more protein will be made; proteins can bind to mRNA and prevent its degradation resulting in protein synthesis Ex. Transferrin – protein receptor that brings iron into a cell Location of the protein Once translated in cytosol – a specific amino acid signal in polypeptide will target protein for its intracellular location Co-translational transfer: proteins destined for lysosomes, ER,, cell membrane or secretion need to be directed to the rough ER for translation Begins on free ribosome in cytosol – signal peptide sequence is translated which binds a signal recognition particle (SRP) Binding of SRP stops translation and directs ribosome to the RER where it binds to a SRP receptor Translation restarts = growing polypeptide moves through a channel into the lumen of RER; once in RER the signal peptide sequence is removed If protein needs to be inserted into a cell membrane = contains a stop transfer sequence When translocator comes into contact with stop transfer sequence translation is paused Translocator discharges the polypeptide into phospholipid bilayer of ER membrane and translation is resumed until polypeptide is complete Destruction of the protein