Gene Expression Notes PDF

Gene Expression (Explaining how cells decode and use the information in their genomes) Gene expression - the flows of genetic information from the genotype to the phenotype DNA RNA Protein One gene - one enzyme (polypeptide) hypothesis (originally proposed by Archibald...

Gene Expression (Explaining how cells decode and use the information in their genomes) Gene expression - the flows of genetic information from the genotype to the phenotype DNA RNA Protein One gene - one enzyme (polypeptide) hypothesis (originally proposed by Archibald Garrod in 1901) – a single gene controlled the synthesis of a single enzyme. Beadle and Tatum (1941) experiment: genetic mutations in the mold Neurospora crassa (mutants/strains of arginine-dependent) Neurospora crassa George W. Beadle Edward L. Tatum The Nobel Prize in Physiology or Medicine in 1958 Gene Expression The Central Dogma is Transcription Translation DNA RNA Protein Transcription - is the process by which the information encoded in DNA is made into a complementary RNA. The segments of DNA transcribed into RNA molecules that can encode proteins are said to produce messenger RNA (mRNA). Other segments of DNA are copied into RNA molecules called non-coding RNAs (ncRNAs). Translation - is the process of synthesizing a specific polypeptide by using the information encoded in the mRNA on a ribosome. Most of genes are transcribed into mRNA, and then the mRNA is translated into polypeptide. Gene expression in prokaryotic and eukaryotic cells Prokaryotic cell Eukaryotic cell Nuclear envelope TRANSCRIPTION DNA DNA TRANSCRIPTION Pre-mRNA mRNA RNA PROCESSING Ribosome TRANSLATION mRNA Polypeptide In eukaryote transcription Ribosome occurs in the nucleus and TRANSLATION In prokaryotes both transcription and translation occurs in translation occur in the cytoplasm due Polypeptide ribosomes present on the to the absence of nucleus rough endoplasmic membrane in the cytoplasm  GENE STRUCTURES : - > PROMOTER > - REGULATORY SEQUENCES TRANSCRIBED REGION > - Transcription > - TERMINATOR A structural gene as a transcriptional unit Gene – an organized unit of DNA sequences that enables a segment of DNA to be transcribed into RNA and ultimately results in the formation of a functional product. A gene is composed of the promoter, the regulatory sequence, the transcribed region and the terminator.  Promoter – the site in the DNA where RNA polymerase binds to begin transcription.  Regulatory sequences – the sites binding to regulatory proteins control whether a gene is turned on or off.  Transcribed region – a region of a DNA that is transcribed into an RNA molecule.  Terminator – a sequence that causes the RNA strand to be released from the transcription complex. Transcription - DNA-directed RNA synthesis (DNA RNA) protein.a Molecular processes: > dependen -  Initiation of transcription requires transcription factors (TFs) binding to the promoter region of a gene.  TFs recruit RNA polymerase binding to a promoter region (with a TATA box in eukaryotes and Pribnow box (TATAAT) in prokaryotes).  Unwinding of the DNA double helix.  In a given region of DNA, only one strand (called the template strand) acts as a template. The DNA template is read from the 3' to 5' direction while RNA is synthesized from the 5' to 3' direction.  Elongation: RNA polymerase catalyzes the transcription, and U rather than T incorporates into RNA.  Transcription ends at a termination site (terminator) (with an AAUAAA signal for polyadenylation). Product: Transcripts - RNAs (mRNA, tRNA, rRNA). TATA- eukaryotic promoter region : RNA polymerase DNA -> redd 3' to S binding region S' to 3 RNA- synthesized TATAAT - > prokaryotic  (3 5) Transcription + DNA = 1 template strand 1 non-template strand (5' % 3) Transcription initiation Transcription elongation Transcription termination I. Rho independent (intrinsic) termination - is when the RNA forms a hairpin structure which displaces RNA polymerase and stops transcription. II. Rho (ATPase) dependent termination - occurs when the rho protein disassociates the RNA polymerase and only I template strand of DNA moves it off of the template. to Synthesize RNA. Transcription Eukaryotic transcription is more complex Location: Prokaryotic - Cytoplasm Eukaryotic - Nucleus Initiation of transcription: Prokaryotic - A single sigma factor Eukaryotic - 5 general transcriptional factors Polymerase: Prokaryotic - A single type of polymerase Eukaryotic - Three types of polymerase (II, I & III) RNA polymerase I transcribes rRNA genes, RNA polymerase II transcribes mRNA, miRNA, snRNA, and snoRNA genes, and RNA polymerase III transcribes tRNA and 5S rRNA genes. RNA I - rRNA genes RNAT-mRNA miRNA , SURNA , , SnORNA RNA-tRNA , SS rRNA Transcription - Eukaryotic RNA processing: A typical eukaryotic Nuclear envelope protein-coding gene has both coding sequences (exons), DNA and non-coding TRANSCRIPTION sequences (internal Pre-mRNA Transcription non-coding sequences – RNA PROCESSING introns, flanking sequences (5′ and 3′ mRNA Capping untranslated regions), a Splicing 5’ cap and a 3’ tail). Tailing Ribosome Exon – a coding portion TRANSLATION of a gene. Polypeptide Intron – a non-coding portion of a gene that is excised from the RNA Eukaryotic cell transcript. Transcription - Eukaryotic RNA processing: RNA splicing – the removal of introns and joining of exons in eukaryotic RNA, forming an mRNA molecule with a continuous coding sequence. Intron RNA is defined by specific sequences within the intron and at the intron-exon boundaries. mRNA splicing occurs in a spliceosome (a large RNA-protein complex) before mature mRNA. tRNAs and rRNAs are self-splicing by a ribozyme. Structure of the human spliceosome prior to exon ligation Transcription - Eukaryotic RNA processing: Biological functions of introns:  Alternate splicing generates different mRNA molecules from the same RNA transcript, thus producing different proteins.  Intron may also contribute to genetic diversity.  Intron may also regulate gene expression. Transcription - Eukaryotic RNA processing: A pre-mRNA processed into mature mRNA by the addition of a 5′ cap, a 3′ poly-A tail Capping – a 7-methylguanosine attached to the mature eukaryotic mRNAs at the 5’ end. (a) Cap structure at the 5’ end of eukaryotic mRNA Tailing – a poly A tail is added to the most mature eukaryotic mRNAs at the 3’ end.  Flanking sequences could regulate the stability of mRNA in the cytoplasm.  They seem to facilitate the export of mRNA. (b) Addition of a poly A tail at the 3’ end of eukaryotic mRNA  They help ribosomes attach to the 5’ end. Transcription Structure of a mature eukaryotic mRNA transcript A fully processed mRNA includes a 5' cap, 5' UTR (untranslated region), coding region (between Start and Stop codons), 3' UTR, and poly(A) tail Protein-coding segment Polyadenylation signal 5’ 5’ Cap 5’ UTR Start codon Stop codon 3’ UTR Poly-A tail Translation The Genetic Code -- the nucleotide information that specifies the amino acid sequence of a polypeptide; the genetic code consists of triplets (codons) of nucleotides. Each codon specifies an amino acid in a polypeptide, or a signal to either start or terminate polypeptides synthesis.  Since there are four bases, there are 64 (43) possible codons.  One start codon (AUG) indicates the starting point of translation, and codes for methionine.  Three stop codes (UAA, UGA, UAG) indicate the end of translation.  60 codons code for particular amino acids.  Met (Start) 1 The Genetic Code: Ala 4 Phe 2 Since there are only 20 Leu 6 Ile 3 different amino acids, Val 4 the genetic code could Trp 1 be redundant Tyr 2 (degeneracy) for certain Ser 6 amino acids, but the Gly 4 code is not ambiguous. Pro 4 Thr 4 His 2 Arg 6 Lys 2 Gln 2 Glu 2 Asn 2 Asp 2 Cys 2 (Stop) 3 The Genetic Code The genetic code is Green Fluorescence Protein (GFP) nearly universal. from Jellyfish Aequorea victoria Genes can be transcribed and translated after being transplanted from one species to another. However, the genetic Recombinant GFP, YFP, CFP, and RFP expressed from E. coli codes of different organisms are often biased towards using one of the several codons that encode the same amino acid over the others - codon usage bias. Genes encoding fluorescent proteins expressed in animals and plants  * IMPORTANT FOR FINAL The Genetic Code Relationships among the coding sequence of a gene, the sequence of a mRNA, the anticodons of tRNA, and the amino acid sequence of a polypeptide Codons must be read in the correct reading frame (correct grouping) in order for the specified polypeptide to be produced. The strand of DNA not used as a template for transcription (non- template strand) is called the coding strand, because it corresponds to the same sequence as the mRNA that will contain the codon sequences necessary to build proteins. The coding strand is also called the sense strand. The coding strand runs in a 5’ to 3’ direction. The DNA strand that mRNA is built from is called the template strand (because it serves as a template for transcription). It is also called the antisense strand. The template strand runs in a 3’ to 5’ direction. Gene mutation In biology, a mutation is the permanent alteration of the nucleotide sequence of the genome of an organism. The most common mutations occur in two ways: 1) a base substitution; 2) an insertion or deletion Common forms of mutations: Base-pair substitution: also called point mutation - the replacement of one nucleotide and its partner in the complementary DNA strand with another pair of nucleotides Missense mutation: a point mutation in which a single nucleotide change results in a codon that codes for a different amino acid. Silent mutation: a mutation where a change in a DNA codon does not result in a change in amino acid specified by the gene. Nonsense mutation: a mutation in which a sense codon that corresponds to one of the twenty amino acids specified by the genetic code is changed to a chain-terminating codon. Frameshift mutation: a mutation caused by insertions or deletions of a number of nucleotides in a DNA sequence that is not divisible by three. Translation Outlines of translation Transcription Translation DNA RNA Protein Translation -- The conversion of Nuclear information provided by mRNA envelope into a specific sequence of amino acids in a polypeptide chain; RNA-directed TRANSCRIPTION DNA polypeptide synthesis (mRNA protein). Pre-mRNA RNA PROCESSING mRNA In prokaryotes, transcription and translation both take place in cytoplasm, while in eukaryotic cells, transcription Ribosome occurs in nucleus and TRANSLATION translation occurs in the Polypeptide cytoplasm. Translation The process requires: tRNA  Amino acids (20 AA) Large subunit Ribosome  ATP and GTP proteins and rRNAs  Enzymes and proteins Small subunit mRNA  RNAs (mRNA, tRNA, rRNA)  Ribosomes (large and small subunits) Schematic model for ribosome structure Translation - tRNA structure  Small RNAs (75-90 bp).  Can base-pair with themselves forming 4- double-helical segments (a cloverleaf pattern).  Anticodon - the three- nucleotide segment that base-pairs with a codon in mRNA.  A free 3’ end single strand region that links to the amino acid corresponding to the anticodon. Secondary structure of tRNA Three-dimensional structure of tRNA Translation - tRNA charge  The process of adding an amino acid to tRNA – also called aminoacylation.  Involves an aminoacyl- tRNA synthetase.  Requires energy (ATP).  Charged tRNA drives the formation of peptide bonds linking amino acids during translation. Translation - Ribosomes  rRNA-protein complexes (ribonucleoprotein particle).  A machinery for carrying out protein synthesis.  Ribosomes are throughout the prokaryotic cells, and in the cytoplasm (freely suspended or attached to ER surface), the nucleolus, and chloroplasts of eukaryotes.  Ribosome is made up of two parts, the large and small subunits.  Function and structure of ribosomes are similar in both prokaryotes and (a) Bacterial ribosomes (b) Eukaryotic ribosomes eukaryotes. “S” for a Svedberg unit - a measure of the sedimentation rate of a particle when centrifuged Translation Polypeptide 50S subunit - Ribosome structure E site (exit site) tRNA Each ribosome has three binding sites for tRNA A site P site E P A (aminoacyl site) (peptidyl site) A site – Aminoacyl- tRNA binding site mRNA 30S subunit P site – Peptidyl-tRNA binding site E site – Exit site 5 3 Schematic model for ribosome structure 50S subunit E site A site P site 30S subunit Bacterial ribosome model based on X-ray diffraction studies Translation - rRNA  Recent research reveals that rRNA, not protein, is 50S subunit primarily responsible for both the structure and function of the ribosome. E site A site  rRNA is the main constituent of interface P site between the two subunits and of the A and P sites.  It is the catalyst of peptide bond formation. 30S subunit  A ribosome can be regarded as one massive ribozyme. Bacterial ribosome model based  rRNAs are highly conserved among all species. on X-ray diffraction studies  mRNA-binding Translation sequence of 16S rRNA Small (30S) ribosomal Small ribosomal subunit - Initiation 1 subunit binds to mRNA.  The initiator Met-tRNA (with GTP bound to Ribosomal-binding 3 5 sequence it) binds to the small ribosomal subunit mRNA and forms a complex. Initiator tRNA  The Met-tRNA/small ribosomal subunit Initiator tRNA 2 binds to start codon in mRNA. complex binds to the 5’ cap of the mRNA. 3 5 Start codon (AUG)  The large ribosomal subunit binds and GTP is hydrolyzed, completing initiation. Large (50S) ribosomal subunit Large 3 ribosomal  Polypeptides initiate with a methionine subunit binds. 70S initiation following a start codon (AUG) and grow complex from the N-terminus towards the C- E site P site terminus along the mRNA in the 5’- to 3’- A site direction. 5 3  tRNA entry: A Translation 1 Charged charged tRNA binds to the A tRNA site. Anticodon - Elongation Polypeptide emerging from exit channel E site A site  mRNA meets the “charged P site tRNAs” entering the A site at a mRNA 3 5 ribosome.  The ribosome travels in 5’ Peptidyl transfer 2 reaction: A bond to 3’ direction and forms between the polypeptide chain and the synthesizes a polypeptide. amino acid in the A site. The polypeptide is transferred to the  The large subunit has A site. 5 3 “peptidyl transferase activity” and catalyzes the reaction. Translocation of 3 ribosome and release of tRNA: The ribosome  The ribosome moves translocates 1 codon to the right. along the mRNA one The uncharged tRNA is released from the E site. codon consisting of 3 This process is repeated again and nucleotides at a time. again until a stop codon is reached. 5 3  Polypeptide A release Translation 1 factor binds to the A site, where a stop codon is found. - Termination Release factor protein  The presence of a stop codon (UAA/UGA/UAG) 5 3 Stop codon in the A site of the Polypeptide The polypeptide ribosome causes 2 is released from the tRNA translation to be in the P site. The tRNA is terminated. then released.  No tRNA has an anticodon that can pair with a stop codon. 5 3 Instead, a release factor The ribosomal 3 subunits, mRNA and binds in the A-site. release factor dissociate.  All of the components disassemble, releasing a mRNA 5 3 50S complete polypeptide. subunit + 30S subunit Transfers of Biological Sequential (Genetic) Information