BISC 101 - Gene Expression & Protein Synthesis PDF

Dr. Onkar S. Bains BISC 101  Central Dogma of Biology Describes the flow of genetic information from DNA to RNA to proteins DNA REPLICATION TRANSCRIPTION NUCLEUS RNA TRANSLATION...

Dr. Onkar S. Bains BISC 101  Central Dogma of Biology Describes the flow of genetic information from DNA to RNA to proteins DNA REPLICATION TRANSCRIPTION NUCLEUS RNA TRANSLATION Protein CYTOPLASM Messenger RNA Used as a template to make 3 to 5% of (mRNA) proteins total RNA Transfer RNA Brings amino acids to mRNA and ~15% of total (tRNA) ribosomes for assembling RNA proteins Ribosomal RNA Makes up structural component ~80% of total (rRNA) of ribosomes RNA Transcription In transcription, a portion of double- stranded DNA template gives rise to a single-stranded RNA molecule – Occurs in nucleus of eukaryotes – Occurs in cytoplasm of prokaryotes (i.e., bacteria) INITIATION STEP: – RNA polymerase binds on the gene's promoter – Promoter: A short DNA sequence having regulatory function over transcription of an adjacent gene, and to which RNA polymerase binds prior to transcription – RNA polymerase binds to promoter with assistance of transcription factors, which are DNA-binding proteins that recognize and accurately initiate transcription at specific promoter sequences Usually found near the beginning of a gene, the promoter has a binding site for RNA polymerase TATA binding protein Typical promoter for RNA polymerase II consists of a TATA box about 25 bases upstream from the startpoint [startpoint designated as +1]  The typical promoter in prokaryotes consists of the start point (designated as +1), the six-nucleotide -10 sequence, and the six-nucleotide -35 sequence – -10 sequence is also known as -10 element (located 10 nucleotides upstream from the startpoint) – -35 sequence is also known as -35 element (located 35 nucleotides upstream from the startpoint) RNA polymerase recognizes and binds directly to these sequences. – The two elements position the RNA polymerase in the right spot to start transcription, and they also make sure this polymerase is pointing in the right direction Image on right from Khan Academy. https://www.khanacademy.org/science/biology/gene-expression-central- dogma/transcription-of-dna-into-rna/a/stages-of-transcription. From “Stages of Transcription”.  ELONGATION STEP: – Synthesis of new RNA strand is 5’ to 3’ (by complementary base pairing) – Reads template strand 3’ to 5’ – As RNA polymerase moves along DNA, it untwists DNA double helix, ~10 to 20 bases at a time – RNA polymerase has a proofreading mechanism that can replace incorrectly incorporated bases 5’ 3’  TERMINATION STEP: – End point of transcription that involves disassembly of RNA polymerase from DNA – RNA polymerase stops when it comes to the termination site on DNA – “Completed” RNA (also known as the primary RNA transcript or pre-RNA) is then subjected to further processing before it leaves the nucleus of the eukaryotic cell  After transcription… In eukaryotes, primary RNA transcript (i.e., pre-RNA) of a gene needs further processing before translation (“RNA processing”) RNA processing involves: 1. Addition of cap to the 5’ end 2. Addition of poly-A tail to the 3’ end 3. Splicing out introns and joining exons In prokaryotes, RNA is ready to be translated into protein – RNA DOES NOT undergo RNA processing  Cap and poly-A tail At 5’ end, a cap is added in the form of 7-methyl guanosine triphosphate At 3’ end, ~100-250 adenine nucleotides are attached (the poly-A tail) Examples of important functions of cap and tail modifications: – The tail facilitates the export of RNA out of the nucleus and into the cell cytoplasm – The cap and tail protect RNA from enzymes (namely RNAses) that can break it down – The cap helps ribosomes attach to the 5’ end of mRNA at the beginning of translation  Introns and exons INTRONS = DOES NOT code for protein (“intervening sequences”) – Intron sequences need to be removed, and remaining ends are re-attached so the final RNA consists of exons only – However, not all eukaryotic RNA transcripts have introns: i.e., histone RNA transcripts lack introns EXONS = codes for protein (“expressing sequences)  Intron splicing Introns are removed from the primary RNA transcript (or pre- RNA) while it is still in the nucleus Introns are “spliced out” by spliceosomes Intron sequences are removed, and remaining ends are re- attached so the final RNA consists of exons only Spliceosome = complex of small nuclear RNAs (snRNAs) and proteins (composed of over 300 proteins)  Central Dogma of Biology Describes the flow of genetic information from DNA to RNA to proteins DNA REPLICATION TRANSCRIPTION NUCLEUS RNA TRANSLATION Protein CYTOPLASM  Translation Process of “decoding” mRNA and using its genetic information to assemble a protein Major players of translation: – mRNA (messenger RNA) – tRNA (transfer RNA) – Ribosomes – Amino acids – the building blocks of a proteins Four phases: activation, initiation, elongation, termination  Messenger RNA (mRNA) Single stranded ribonucleotides that is complementary to its DNA gene template Takes instructions from DNA in the nucleus (transcription) to the ribosomes in cytoplasm mRNA is "read" according to the genetic code, which relates DNA sequence to amino acid sequence in proteins ― mRNA is read in a series of triplets (3 nucleotides) called codons Each codon specifies a particular amino acid that is to be placed in polypeptide chain for construction of protein  Genetic Code Code is almost “universal” – used by both prokaryotes and eukaryotes Each group of 3 nucleotides on the mRNA is a codon 64 possible codons, but code for 20 different amino acids More than one codon is used for most amino acids: the genetic code is “degenerate” – This means that it is not possible to take a protein sequence and deduce exactly the base sequence of the gene it came from In most cases, third base of the codon (wobble base) can be altered without changing the amino acid 61 codons code for amino acids – 5'-AUG-3' is used as start codon (codes for methionine) 3 codons are stop codons (5'-UAA-3', 5'-UAG-3', 5'-UGA-3') – Translation ends at a stop codon which codes for no amino acid 5'– First base – Second base – Third base –3'  You should be able to use the codon table, and determine the amino acid sequence from either mRNA or even DNA  Transfer RNA (tRNA) Transfer RNA are short RNA sequences that fold into a characteristic cloverleaf pattern Each tRNA has 3 bases that make up the anticodon – These bases pair with the 3 bases of the codon on mRNA during translation Hydrogen Each tRNA has its corresponding bonding amino acid attached to the 3’ between base pairs end (part of activation step of translation)  Ribosomes “Protein factory” of cells made up of two subunits: large and small The P site (peptidyl-tRNA site) holds tRNA with newly forming polypeptide chain attached The A site (aminoacyl-tRNA site) holds tRNA carrying next amino acid to be added to polypeptide chain The E site (exit site) holds free tRNA before it is released from ribosome/RNA template translation complex Ribosome  Initiation Initiation stage brings together mRNA, a tRNA carrying the first amino acid (methionine), and two ribosomal subunits With assistance of special proteins called initiation factors, the small ribosomal subunit binds near 5’ end of mRNA (more specifically it binds to the ribosomal binding site on mRNA) – Ribosomal binding site = sequence of nucleotides upstream from mRNA start codon Afterwards, tRNA carrying methionine binds to start codon (5’-AUG-3’) on mRNA The large subunit is brought in next with help from an energy-rich molecule GTP in order to complete the whole initiation complex Ribosome  Elongation Elongation begins with binding of tRNA carrying an amino acid to A site (this step is catalyzed by GTP and special proteins called elongation factors) The free tRNA leaves the E site Ribosome moves Once incoming tRNA downstream along mRNA binds to A site, the to next codon polypeptide chain is (translocation is promoted moved from tRNA in P by elongation factors and site to amino acid GTP) attached to tRNA in A site  Termination When translocation opens the A site and exposes one of the stop codons, a protein called a release factor fills the A site Release factors bind to codon - there are no tRNA molecules with anticodons for stop factors Release factor binding triggers polypeptide chain to be cleaved from tRNA Polypeptide is released, and then the tRNA is released The two ribosomal subunits and mRNA dissociate from each other (catalyzed by GTP) Transcription and translation are coupled and occur simultaneously in bacteria REMEMBER - eukaryotic transcription and translation are spatially and temporally isolated  Monocistronic vs. polycistronic mRNA Monocistronic – One mRNA molecule translates for only one type of protein – Refers to eukaryotic mRNA Polycistronic – One mRNA molecule translates for more than one type of protein – Refers to prokaryotic mRNA