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Transcription and Translation Genes The structure of a prokaryote gene Gene • Consensus sequences • - 10 and -25 upstream from the start • 3’ TAC – start or initiator codon • Establishes the reading frame – ORF – open reading frame • Bases grouped in 3’s triplet code • Proceeds to stop ( ATT , ATC, ACT or termination sequence in prokaryotes The Pribnow Box and Shane -Dalgarno • The RNA binding site has a consensus sequence of 5’ TATAAT 3’ ( -) and 3’ ATATTA 5’ (+) • This is where the DNA begins to become unwound for transcription • The initially transcribed sequence of the gene may not reflect doing but may be a leader sequence. • The prokaryotes usually contain a consensus sequence known as the Shane Delgarno which is complememtary to the 16s rRNA on the ribosome ( small subunit ) • The leader sequence also may regulate transcription Promoters are at the beginning of the Gene • RNA polymerase recognizes a binding site in front of the • • • 5’ gene. This is referred to as upstream of the gene. The direction of transcription is referred to as downstream Different genes have different promoters. IN E. coli the promoters have two functions The RNA recognition site for transcription which is the consensus sequence for prokaryotes is TTGACA3’ ( Watson strand) which means on the reading strand 3’ AACTGT5’ ( Crick strand) Genes and Gene Expression • Genes are written in a code consisting of groups of three letters called triplets. • There are four letters in the DNA alphabet. There are 64 possible arrangements of the four letters in groups of three • The triplets specify amino acids for the synthesis of proteins from the information contained in the gene Genes - Continued • Genes can also specify t- RNA or r- RNAs • The gene begins with a start triplet and ends with a • • • • stop. The bases between the start and the stop are called an open reading frame, ORF. The information in the gene is transcribed by RNA polymerase. It reads the gene from 3’ to 5’ The template strand is now referred to as the CRICK strand and the nontemplate strand is now known as the WATSON strand DNA sequences are stored in data bases as the WATSON strand Reference - COLD SPRING HARBOR - 2003 Prokaryote Genes are • Continuous • They do not contain introns like eukaryote genes • The gene consists of codons that will determine the sequence of amino acids in the protein • At the end of the gene there is a terminator sequence rather than an actual stop • The terminator may be at the end of a trailer sequence located downstream from the actual coding region of the gene Transcription Begins • DNA is read 3’ to 5’ and m RNA is synthesized 5’ to 3’ • 3’ TAC is the start triplet • This produces a complementary mRNA message 5’ AUG 3’ – • Groups of three bases in the messenger RNA formed are referred to as CODONS RNA POLYMERASE RNA Polymerase • There are approximately 2000 molecules of RNA Polymerase per bacterial cell • E. coli RNA polymerase is composed of six subunits with a molecular weight of over 400,000 Da RNA POLYMERASE • Enzyme has been described as a claw • The Beta portions of the claw form the • • pincers The Alpha portion are on the other end of the claw The Omega portion is wrapped around the Beta portion RNA Polymerase Action • Does not require a primer in comparison to DNA Polymerase • Binds to the promoter sequence and opens the double strands of DNA • Builds a chain of RNA by connecting the 5’ end of a nucleotide to the 3’ end of the nucleotide in front of it Language • RNA polymerase utilizes the sequence of the template strand in DNA language to build a complementary copy of RNA • The strand that is the template is referred to as the transcribed strand • The strand with the same sequence as RNA with the exception of U instead of T is referred to as the coding strand RNA Polymerase Structure – Core enzyme Polymerase sub units • α2: the two α subunits assemble the enzyme and • • • • recognize regulatory factors. These compose the core enzyme β: this has the polymerase activity (catalyzes the synthesis of RNA) which includes chain initiation and elongation. β': binds to DNA (nonspecifically). ω: restores denatured RNA polymerase to its functional form in vitro. It has been observed to offer a protective/chaperone function to the β' subunit RNA Polymerase Holoenzyme Sigma Factors • A sigma factor (σ factor) are proteins that are required for the binding of the RNA polymerase to the promoter so that transcription can be initiated. They are also active in the elongation process. Transcription and the Initiation Complex • The core enzyme binds to the holoenzyme • This is called promoter recognition • The sigma factors first bins to the -10 region • When the Beta pincer closes around the DNAto form the active site channel around the DNA, this opens the strands Rifampin • The initiation somplex can be blocked by the antibiotic Rifampin • Rifampin binds to the RNA Polymerase at the active site channel is such a way that it blocks the elongation of the RNA Mutations, Antibiotics, and Antibiotic Resistance Initiation, Elongation, Termination Transcriptional overview • http://vcell.ndsu.nodak.edu/~christjo/vcell /animationSite/transcription/elongation.ht m Rho independent( Factor dependent) • The termination site consists of two sequences • The first is an inverted repeat • When the inverted repeat is transcribed it forms a hairpin • The inverted repeat is followed by a string of AAAAAAAAAAAs • The RNA molecule will have terminal UUUUs • AU pairs are less stable and this causes the release of the RNA molecule from the DNA Termination- Factor Independent Factor Dependent • Rho Dependent ( Rho the most universal factor in prokaryote cells). There are other factors • Rho functions in the synthesis of RNAs that are not being translated • Rho forms a ring that binds to a sequence in the RNA called the rut site • Binds to the RNA and affects the action of RNA polymerase Eukaryote Transcription and Transcription Factors Genes for t RNAs and r RNAs • The genes for t RNAs have a promoter and transcribed leader and trailer sequence that are removed prior to their utilization in translation. Genes coding for tRNA may code for more than a single tRNA molecule • The segments coding for r RNAs are separated by spacer sequences that are removed after transcription. • The acceptor stem • • • • includes the 5' and 3' ends of the tRNA. The 5' end is generated by RNase P The 3' end is the site which is charged with amino acids for translation. Aminoacyl tRNA synthetases interact with both the acceptor 3' end and the anticodon when charging tRNAs. The anticodon matches the codon on mRNA and is read 3’ to 5’ t-RNA t- RNA • Found in the cytoplasm • Amino acyl t- RNA synthetase is an enzyme that enables the amino acid to attach to t-RNA • Also activates the t- RNA • Clover leaf has a stem for attachment to the amino acid and an anticodon on the bottom of the clover leaf t- RNA Common Features • a CCA trinucleotide at the 3' end, unpaired • four base-paired stems, and • One loop containing a T-pseudoU-C sequence and another containing dihydroU. tRNA • tRNAs attach to a • • • • specific amino acid and carry it to the ribosome There are 20 amino acids 61 different codons for these amino acids and 61 tRNAs The anticodon is complementary to the codon Binds to the codon with hydrogen bonds Ribosomal genes • Very similar to the structure of protein genes tRNA and rRNA genes • The genes for rRNA are also similar to the organization of genes coding for proteins • All rRNA genes are transcribed as a large precursor molecule that is edited by ribonucleases after transcription to yield the final r RNA products Ribosomal RNA • Combines with specific proteins to form ribosomes • Serves as a site for protein synthesis • Associated enzymes and factors control the process of translation Prokaryote Ribosomes • Ribosomes are small, but complex structures, roughly 20 to 30 nm in diameter, consisting of two unequally sized subunits, referred to as large and small which fit closely together as seen below. • A subunit is composed of a complex between RNA molecules and proteins; each subunit contains at least one ribosomal RNA (rRNA) subunit and a large quantity of ribosomal proteins. • The subunits together contain up to 82 specific proteins assembled in a precise sequence. 30s and 50s Ribosomal subunits Prokaryote ribosomal RNA Type of rRNA Approxim ate number of nucleotid es Subunit Location 16s 1,542 30s 5s 120 50s 23s 2,904 50s Prokaryote ribosomes – polysomesthe process of translation Prokaryote transcription and translation • Prokaryote transcription and translation take place in the cytoplasm • All necessary enzymes and molecules are present for the transcription and translation to take place Translation • A molecule of messenger RNA binds to the 30S ribosome ( small ribosomal unit) at the Shine Dalgarno sequence • This insures the correct orientation for the molecule • The large ribosomal sub unit locks on top T RNA and Amino acyl t RNA synthetase( Preparation of tRNAs) • The enzyme amino acyl t RNA Synthetase connects the amino acid to its specific t RNA to form cognate t RNA • Hydrolyzes ATP to transfer the amino acid to the 3’OH end of the t RNA • The acceptor arm of the t RNA ends in the sequence CCA The Ribosome • There are four significant positions on the ribosome • EPAT • When the 5’ AUG 3’ of the mRNA is on the P site the t-RNA with the anticodon, 5’UAG3’ forms a temporary bond to begin translation Ribosomal Sites • T site – the 5’ end of the messenger RNA • • • enters the ribosome A site – acceptor site -the relationship of the mRNA to the ribosome is stabilized at this site – the amino acylated t RNA is bound to the mRNA P site the peptide bonds are formed between the amino acids on the t RNAs E site - The mRNA moves to the final position on the ribosome as the tRNA is released EPAT Ribosomal structure Translation Initiation Region • Should contain the initiator codon( start) AUG, may also be GUG • This may be at the start of the gene or in an Untranslated leader sequence upstream of Gene ( UTR) • Shine Delgarno named for two scientists who discovered it at -35 binds to the 30s ribosomal subuit ( 16srRNA) Archaea Initiation • Combination of Eukarya and Bacteria Initiation • Archaea and Eukarya have more initiation factors than Bacteria • Archaea uses formylated methionine Initiation Elongation Elongation Translocase • Moves the tRNA from the A site to the P site • This displaces the t RNA on the P site to the E site • Elongation factors function at this point • Energy required from the hydrolysis of GTP Peptidyl transferase • Forms the peptide bonds between the amino acids attached to the t RNAs • Forms the nascent polypeptide chain Termination • http://www.phschool.com/science/biology _place/biocoach/translation/term.html • Release factors function in termination ( RF1 and 3) • GTP is required for energy • The termination or stop codon enters the A site( UAA, UAG, UGA) • Recognized by the ribosome Termination From Gene to polypeptide Codon chart •There is wobble in the DNA code – This is a protection from mutations •More than one codon can specify the same amino acid • Note arginine - CGU, CGC,CGA, CGG all code for arginine – only the third base in the codon changes •There are two additional codons for arginine as well AGA and AGG these reflect the degenerate nature of the code WOBBLE E. Coli Gene Map Mutations in DNA • May be characterized by their genotypic or phenotypic change • Mutations can alter the phenotype of a microorganisms in different ways • Mutations can involve a change in the cellular or colonial morphology Types of Mutations • Conditional mutations are those mutations that are expressed only under specific environmental conditions ( temperature) • Biochemical mutations are those that can cause a change in the biochemistry of the cell ( these may inactivate a biochemical pathway) • These mutants are referred to as auxotrophs because they cannot grow on minimal media • Prototrophs are usually wild type strains capable of growing on minimal media Two types of mutations • Spontaneous mutations – These occur without • • a causative agent during replication Induced mutations are the result of a substance referred to as a mutagen Cairns reports that a mutant E. coli strain unable to use lactose is able to regain its ability to use the sugar again – should this be referred to as adaptive mutation? Hypermutation • One possible explanation is hypermutation • A starving bacterium has the ability to generate • multiple mutations with special mutator genes that enable them to form bacteria with the ability to metabolize lactose This is an interesting theory still under investigation Spontaneous mutations Types 1. A purine substitutes for a purine or a pyrimidine substitutes of a pyrimidine. This type of mutation is referred ta as a transition. Most of these can be repaired by proofreading mechanisms 2. A pyrimidine substituted for by a purine is referred to as a transversion. These are rarer due to steric problems in the DNA molecule such as pairing purines with purines. 3. Insertions or deletions cause frame shifts – the code shifts over the number of bases inserted or deleted Mutation Types • Errors in replication due • • to base tautomerization AT and CG pairs are formed when keto groups participate in hydrogen bonds In contrast enol tautomers produce AC and GT base pairing Spontaneous mutations – another cause • • • • Depurination A purine nucleotide can lose its base It will not base pair normally It will probably lead to a transition type mutation after the next round of replication. • Cytosine can be deaminated to uracil which can then create a problem Frame Shifts • Additions and deletions change the reading frame. • The hypothetical origin of deletions and insertions may occur during replication • If the new strand slips an insertion or addition may occur • If the parental slips a deletion may occur Mutagenesis • a. b. c. d. Any agent that directly damages DNA, alters its chemistry, or interferes with repair mechanisms will induce mutations Base analogs Specific mispairing Intercalating agents Ionizing radiation Base analogs are structurally similar to normal nitrogenous bases and can be incorporated into the growing polynucleotide chain during replication. The expression of mutations • Forward mutations – a mutation from the • • • wild type to a mutant form is called a forward mutation Reversion-If the organism regains its wild type characteristics through a second mutation Back mutation – The actual nucleotide sequence is converted back to the original Suppressor mutation – overcomes the effects of the first mutation More on mutations • Point mutations – caused by the change in one • • • DNA base Silent mutations – mutations can occur which cause no effect – this is due to the degeneracy of the code ( more than one base coding for the same amino acid) Missense mutation – changes a codon for one amino acid into a codon for another amino acid Nonsense – In eukaryotes the substitution of a stop into the sequence of a normal gene Detection and isolation of mutants • • • • • • Requires a sensitive system Mutations are rare One in about every 107 – 1011 Replica plating is a technique that is used to detect auxotrophs It distinguishes between wild type and mutants because of their ability to grow in the absence of a particular biosynthetic end product Replica plating allows plating on minimal media and enriched media from the same master plate The selection of auxotorph revertants • The lysine auxotrophs ( Lys-) are treated with a mutagen such as nitroquanidine or uv light to produce revertants Ames Test • Developed by Bruce Ames • Used to test for carcinogens • A mutational reversion assay based upon mutants of Salmonella typhimurium DNA repair mechanisms Type I -Excision repair Corrects damage which causes distortions in the double helix • A repair endonuclease or uvr ABC endonuclease removes the damaged bases along with some bases on either side of thee lesion • The usual gap is about 12 nucleotides long. It is filled by DNA polymerase and ligase joins the fragments. • This can remove Thymine-Thymine dimers • A special type of repair utilizes glycosylases to remove damaged or unnatural bases yielding the results discussed above Mutations and repair Type II – Removal of lesion • Thymine dimers and alkylated bases are often repaired directly • Photoreactivation is the repair of thymine dimers by splitting them apart into separate thymines with the aid of visible light in a photochemical reaction catalyzed by the enzyme photolyase • Light repair -phr gene - codes for deoxyribodipyrimidine photolyase that, with cofactor folic acid, binds in dark to T dimer. When light shines on cell, folic acid absorbs the light and uses the energy to break bond of T dimer; photolyase then falls off DNA Dark repair of mutations • Dark repair Three types 1) UV Damage Repair (also called NER - nucleotide excision repair) Excinuclease (an endonuclease; also called correndonuclease [correction endo.]) that can detect T dimer, nicks DNA strand on 5' end of dimer (composed of subunits coded by uvrA, uvrB and uvrC genes). UvrA protein and ATP bind to DNA at the distortion. UvrB binds to the UvrA-DNA complex and increases specificity of UvrA-ATP complex for irradiated DNA. UvrC nicks DNA 8 bases upstream and 4 or 5 bases downstream of dimer. UvrD (DNA helicase II; same as DnaB used during replication initiation) separates strands to release 12-bp segment. DNA polymerase I now fills in gap in 5'>3' direction and ligase seals. The Effects of uv light Post replication repair • If T dimer not repaired, DNA Pol III can't make • • • complementary strand during replication. Postdimer initiation - skips over lesion and leaves large gap (800 bases). Gap may be repaired by enzymes in recombination system - lesion remains but get intact double helix. Successful post replication depends upon the ability to recognize the old and newly replicated DNA strands This is possible because the newly replicated DNA strand lack methyl groups on their bases, whereas the older DNA has methyl groups on the bases of both strands. The DNA repair system cuts out the mismatch from the non- methylated strand Recombination repair • The DNA repair for which there is no remaining template is • • • • restored RecA protein cuts a piece of template DNA from a sister molecule and puts it into the gap or uses it to replace a damaged strand Rec A also participates in a type of inducible repair known as SOS repair. If the DNA damage is so great that synthesis stops completely leaving many gaps, the Rec A will bind to the gaps and initiate strand exchange. It takes on a proteolytic funtion that destroys the lexA repressor protein which regulates genes involved in DNA repair and synthesis