Gene

Gene - DNA structure o A=T (2H bond) o G=T (3H bond) - Purine pair with pyrimidine - Exist as right handed helix, looks counter clockwise when viewed from above - 10 base pairs per turn of helix - Consecutive bases are separated from each by 0.34 nm - Pitch Æ vertical height of 1 turn of double helix - 1 complete turn of helix = 3.4nm/turn - Each bp rotated 36 degrees related to its immediate neighbors - Strands run anti-parallel directions o Strand 1 runs 5’ to 3’ o Strand 2 runs 3’to 5’ o 5’Æ phosphate , 3’Æ hydroxyl RNA structure - Usually single stranded - Uracil (U) instead of Thymine (T) Types of RNA 1. 5 (or more) types a. Ribosomal RNA b. Transfer RNA c. Messenger RNA d. RNA enzymes e. Other stable RNA’s (gene regulation) 2. Different from each other in function, site of synthesis in eukaryotic cell and structute. Organization of DNA in cells • Procaryotes o Circular chromosome associated with basic protein to aid packaging • Eukaryotes o Linear chromosome associated with basic protein known as Histone o Histones coils DNA into nucleosome o Nucleosome form chromatin DNA replication • Process of copying genetic information in parental DNA • Objective ; transmission of genetic information to offspring • Semi-conservative o One strand from parent in each of new double helices • Each parental strand is conserved • Two parental strand separate and serve as template for synthesis of new strand • Replication is initiated at precise point of the chromosomeÆ origin of replication

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Active DNA replication occurs at the Replication Forks (these arise from the origin of replication) Replication fork is dynamic (it moves) the origin of replication is static Replication is bidirectional from a single origin ; replication forks move in opposite direction from the origin

Prokaryotes - Bidirectional - Single origin of replication Eukaryotes - Bidirectional - Multiple origin of replication

Mechanism of DNA replication Involves many enzymes and protein Helicase Æ unwind the helix - Once strand separated, need to keep them apart Æ single stranded binding protein (SSBs) o Keep strand apart but not cover up the bases - Unzipping introduces tension and supercoil in helix Æ tension relieved by cutting and religation of backbone by Topoisomerase e.g. (DNA ligase) DNA polymeraseÆ catalyzed new formation of DNA strand DNA polymerase III synthesis DNA Æ from 5’to 3’ - New DNA strand synthesize in 5’to 3’ direction o New nucleotide added to 3’ end of growing chain - Template read 3’ to 5’ direction - DNA polymerase does not initiate replication spontaneously - New nucleotide add to RNA oligonucleotide primer - Primer made by primase - RNA primer is eventually removed and replace by DNA - The process of replicating both strand involves a “Leading” and “Lagging” strand - Leading strand synthesis is continuous - Lagging strand synthesis is discontinuous o Made in short fragment = okazaki fragment o Joined to each other by DNA ligase - DNA polymerase I remove primers and fill gaps - DNA ligase joints fragments to form complete strands of DNA

Genetic code - Sequence of base in DNA correspond to amino acid sequence of polypeptide encoded 20 amino acid in protein Nirenberg and Khorana • Discovery of codonÆ 3 nucleotides per amino acid • The genetic code is degenerate more than one codon for some amino acid • Codons are translated by anti-codons • Nucleotide 1 and 2 of codon are most important in specifying amino acids • Wobble hypothesis; nucleotide 3 is not as critical • Cell do not make tRNAs to recognize all codons

Gene structure Gene - Polynucleotide sequence (DNA or RNA) that are transcribed (made into RNA) - CISTRON Æ another name of gene - May encode mRNA, tRNA, rRNA etc. o Final product gene not always protein - Organization of genes on chromosome - Some bacteria and some viruses have overlapping gene - Template strand : DNA strand that contain coding information and directs RNA synthesis - Non-template strand ; non-coding DNA strand - Transcription Æ synthesis of RNA from DNA sequence - Key enzyme Æ RNA polymerase o Promoter serve as recognition and binding site for RNA polymerase - Sequence is encoded starring at 3’ end of the template strand (DNA) - Gene orientation described with respect to RNA o 5’ end made first Æ gene begin at 5’ and end at 3’ -

+1 signifies start of transcription Downstream of promoter Upstream of translation start site

Leader sequence - Between +1 and start codon - Usually not translated Shine-Dalgarno sequence; Ribosome binding site (RBS) - Contain in leader sequence - Helps define start site of translation Coding regionÆ region of gene that specifies the amino acid sequence of a protein Trailer = region at 3’ end of gene that is transcribed but not translated - Important for regulation by stability

Terminator - Signal end of transcription - Txn process beyond stop codons in coding region Coding region begins with translation initiation codon; • Starts with the sequence 5’-AUG-3’ on mRNA • Complementary to 3’-TAC-5’ on template strand of DNA coding region end at stop codon. • mRNA in bacteria may be mono-cistronic or polycistronic • in eukaryotes mRNA are monocistronic o monocistronic ; 1 gene per transcript o polycistronic ; >1 genes per transcript tRNA and rRNA gene - Generally; polycistronic in both eukaryotes and prokaryotes - Individual rRNAs and tRNAs subsequently generated by Post-transcriptional cleavages - rRNA genes have promoter, leader, coding, spacer and trailer region. - Spacer and trailer region may encode tRNA molecules

Polycistronic - tRNA genes have promoter, leader, spacer, trailer regions - leader, spacer, trailer removed during maturation process Prokaryotic vs. eukaryotic genes - prokaryotes (and viruses) o coding information is usually continuous - eukaryotes o most gene have coding sequences interrupted by non-coding sequences o exon = coding sequence o intron = non-coding sequence o introns removed by spilcing Transcription Process of converting DNA into RNA : could be mRNA, tRNA, rRNA or other RNA form Transcription in bacteria • mRNA may be mono or poly – cistronic • polycistronic mRNA are not processed o each has its own ribosome binding site Transcription in prokaryotes (bacteria) - catalyzed by RNA polymerase (a single RNA polymerase) - large multi subunits enzyme

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core enzyme = a2BB holoenzyme = core enzyme+sigma factor ( direct core enzyme to promoter)

Key Step in Transcription 1) Initiation a. Binding of RNA polymerase to the promoter b. Initial synthesis of RNA strand ~first 10 nucleotides 2) Elongation a. Continued synthesis of RNA strand at 3’end of gene b. RNA strand is made 5’to 3’ 3) Termination a. Termination of synthesis of RNA strand at 3’ end of gene b. Dissociation of RNA polymerase from DNA Initiation - Sigma factor / subunit needed for initiation - >1 sigma factor in the cell - Allow RNA polymerase to identify promoters - Sigma factor only necessary at the initiation of transcription and it dissociates during elongation - Is not always associated with RNA polymerase Elongation - Continued synthesis of RNA strand - RNA strand is made 5’to 3’ - Sigma factor not needed for elongation Termination - Synthesis continues until terminator reach - Release of RNA polymerase and RNA transcript

Transcription in eukaryotes - 3 major RNA polymerase in eukaryotes o RNA polymerase I makes rRNA o RNA polymerase II makes mRNA o RNA polymerase III makes tRNA - Instead of sigma factor eukaryotes use TRANSCRIPTION FACTOR Maturation of eukaryotic mRNA - Initial mRNA transcriptÆ heterogenous nuclear RNA (hnRNA) - Located in nucleus - Processed to final mRNA product located in cytoplasm

Maturation of mRNA Æ only in eukaryotes 1. Synthesis of transcript = hnRNA 2. Addition of 5’ cap = 7 methyl guanosine 3. Removal of intron and spilicing of exon by spliceosome a. Exon (expressed region) Æ present in final mRNA product b. Intron (intervening region) Æ not present in final mRNA product 4. Addition of 3’ poly (A) tail RNA polymerase transcribed gene on both strand of DNA on a chromosome Different gene may not share the same strand of DNA as the template strand Prokaryotes mRNA does not have 5’cap, introns, exons and 3’poly (A) tail

Translation - Conversion of mRNA to protein Protein synthesis 1) Ribosome a. Site of translation 2) Single mRNA can be translated by >1 ribosome a. Polysome or polyribosome; mRNA complex with several ribosomes 3) Direction of protein synthesis; N to C terminal a. Ribosome move on mRNA in 5’ to 3’ direction 4) Transcription and translation occur simultaneously in prokaryotes but not common in eukaryotes Ribosome - Consist of protein and rRNA - Translation take place in a groove between large and small subunits - rRNA important for o structure of ribosome o determining site of translation initiation in prokaryotes o catalyctic role in protein synthesis Steps in protein synthesis 1. Amino Acid activation 2. Initiation 3. Elongation 4. Termination tRNA - Acceptor arm Æ 3’end of tRNA “holding” activated amino acid

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Anticodon loop Æ contain triplet of nucleotide (anticodon) are complimentary to the codon on mRNA

Transfer RNA and amino acid activation - Important that the right tRNA is connected to the right amino acid - Catalyzed aminoacyl-tRNA synthase o At least 20 amino acid o Specifically bind to amino acid and cognate tRNA’s - Amino acyl tRNA synthase have proofreading activity correct mischarging erros. 1. Amino acid activation - Attachment of amino acid to tRNA - Produce protein 2. Initiation a. Starts when initiator methionine (tRNA-Met) recognize AUG b. Recognition involves between RBS and 16rRNA c. Formation of complex between mRNA, initiator tRNA large and small ribosomal subunits d. Requires initiation factors (proteins) and energy (GTP)

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Initiation of translation The goal Æ position ribosome properly at 5’end of mRNA Initiator tRNA recognize AUG RBS Æ 16 rRNA interaction complex with mRNA, initiator tRNA, ribosomal subunits Requires initiation factors and energy (GTP)

3. Elongation of polypeptide chain - Sequential addition of amino acids to growing polypeptide chain - 3 phases o Aminoacyl – tRNA binding o Transpeptidation reaction (addition next amino acid) o Translocation - Involves several elongation factors - Require energy GTP

tRNA binding site of ribosome - Peptidyl (donor; P) site o Binds initiator tRNA or tRNA attach to growing polypeptide (peptidyltRNA) - Aminoacyl (acceptor ; A) site o Binds incoming aminoacyl-tRNA - Exit (E) site o Briefly bind empty tRNA before it leaves Ribosome

Elongation - Aminoacyl-tRNA binding to A site of Ribosome - Transpeptidation Æ transfer of peptide chain from peptidyl-tRNA in P site to aminoacyl tRNA in A site catalyzed by Peptidyl transferase - Translocation Æ movement of ribosome to new codon o tRNA in A site enters P site o tRNA in P site enters E site (uncharged tRNA) o A site empty – ready for next aminoacyl tRNA 4. -

Termination Occurs at stop codons Require release factors and energy GTP Cleavage of complete polypeptide chain from tRNA also catalyzed by peptidyl transferase

Gene Regulation - Level of regulation of gene expression - Occurs at many levels o Transcription initiation o Transcription elongation o Translation o Post translation Regulation of Transcription initiation (mRNA synthesis regulation) - Objective Æ produce appropriate mRNA that encode enzymes in response to change in environment - Slower than direct regulation enzyme activity but more efficient Î controls the amount of enzyme product General concept of gene regulation 1. Transcription of genes can be induced (activate) or repressed (turn off) a. Generally presence or absence of substrate in environment determines gene expression 2. Gene that are repressed are subjected to negative control 3. Gene that are induced are subjected to positive control 4. Regulation protein mediate positive and negative control a. Repressor block / activator enhance 5. Regulatory protein exist in 2 state a. Active and inactive depending on presence or absence of inducing or repressing molecule.