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Biology Chapter 16.docx

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McMaster University
Lovaye Kajiura

Biology Chapter 16: Transcription and Translation Bernard Ho November 27, 2010 Transcription in Bacteria − The first step in converting genetic information into proteins is the synthesis of a messenger RNA version of the instructions archived in DNA − Enzymes called RNA polymerases are responsible for synthesizing mRNA − Like DNA polymerases, an RNA polymerase performs a template-directed synthesis in the 5’  3’ direction − Unlike DNA polymerases, RNA polymerases do not require a primer to begin transcription − Transcription occurs when RNA polymerase matches the base in a ribonucleotides triphosphate with the complementary base in a gene (a section of DNA that codes for a protein or RNA) − Once a matching ribonucleotides is in place, RNA polymerase catalyzes the formation of a phosphodiester bond between the 3’ end of the growing mRNA chain and the new ribonucleotides − As this matching-and-catalysis process continues, an RNA that is complementary to the gene is synthesized − Only one of the two DNA strands is used as a template and transcribed, called the template strand − The other strand is called the non-template strand, also known as the coding strand because its sequence matches the sequence of RNA that is transcribed from the template strand and codes for a polypeptide − Key difference is that RNA has uracil (U) rather than thymine (T) − RNA Polymerase Structure and Function o To understand structure, biologists used X-ray crystallography, which allowed them to obtain information about the 3D structure o Results indicate that the enzyme is large and globular and has several prominent channels running through the interior o The enzyme’s active site, where the phosphodiester bonds form, is located where several of these channels intersect − Initiation: How Does Transcription Begin o Researchers discovered that the enzyme cannot initiate transcription on its own o Instead, a detachable protein subunit called sigma must bind to RNA polymerase before transcription can begin  Sigma is a protein that recognizes promoter regions o RNA polymerase and sigma form a holoenzyme, which consists of a core enzyme, which contains the active site for catalysis, and other required proteins o When researchers mixed RNA polymerase, sigma and DNA together, they found that the holoenzymes bound tightly to specific sections of DNA o These binding sites were named promoters because they are sections of DNA where transcription begins o The discovery of promoters suggests that sigma’s function is regulatory in nature o Sigma appeared to be responsible for guiding the RNA polymerase to specific locations where transcription should begin o Results showed that promoters were located on the non-template strand, were 40-50 bp long and had a particular section that looked similar o This similar segment of DNA had a series of bases identical or similar to TATAAT o This six base pair sequence is now known as the -10 box because it is centred about 10 bases from the point where RNA polymerase starts transcription o DNA that is located in the direction RNA polymerase moves during transcription is said to be downstream o DNA in the opposite direction is said to be upstream, thus the -10 box is centred 10 bases upstream from the transcription start site o The place where transcription begins is the +1 site o Researchers also recognized that the sequence TTGACA occurred in these same promoters, centred about 35 bases upstream from the +1 site o This second key sequence is called the -35 box o Follow up work showed that transcription begins when sigma binds to the -35 and -10 boxes o Supports the hypothesis that sigma is a regulatory protein o Sigma tells RNA polymerase where and when to start synthesizing RNA o Once sigma binds to a promoter, the DNA helix opens and creates two strands of single-stranded DNA o The template strand is threaded through a channel that leads to the active site inside RNA polymerase o Monomers known as ribonucleoside triphosphate (NTPs) enter a channel at the bottom of the enzyme and diffuse to the active site o When an incoming NTP pairs with a complementary base on the template strand of DNA, RNA polymerization begins o Sigma is released once RNA synthesis is under way − Elongation o During the elongation phase of transcription, RNA polymerase moves along the DNA template strand in the 3’  5’ direction of the template strand, synthesizing RNA in the 5’  3’ direction o In the interior of the enzyme, a group of projecting AA called the enzyme’s zipper helps open the double helix at the upstream end and a nearby group of AA called the rudder steers the template and non-template strands through channels inside the enzyme o Meanwhile, the enzyme’s active site catalyzes the addition of nucleotides to the 3’ end of the growing RNA molecule at the rate of about 50 nucleotides/second o During elongation, all the prominent channels or grooves in the enzyme are filled o Double stranded DNA goes into and out of one groove, NTPs enter another and the growing RNA strand exits to the rear − Termination o In most cases, transcription stops when RNA polymerase reaches a stretch of DNA sequence that functions as a transcription termination signal o The bases that make up a termination signal code for a stretch of RNA with an unusual property, as soon as it is synthesized, the RNA sequence folds back on itself and forms a short double helix that is held together by complementary base pairing o The secondary structure that results is called a hairpin o The formation of the hairpin structure is thought to disrupt the interaction between RNA polymerase and the RNA transcript, resulting in the physical separation of the enzyme and its product − Summary o Transcription begins when sigma, as part of the holoenzyme complex, binds to the promoter at the start of a gene o Once binding occurs, RNA polymerase begins to synthesize mRNA by adding ribonucleotides that are complementary to the template strand in DNA o Transcription ends when a termination signal at the end of the gene leads to the formation of hairpin in mRNA, disrupting the transcription complex Transcription and RNA Processing in Eukaryotes − Transcription in eukaryotes similar to bacterial transcription in that RNA polymerase does not bind directly to promoter sequences by itself − Instead, proteins called basal transcription factors initiate eukaryotic transcription by matching the enzyme with the appropriate promoter region in DNA − The function of basal transcription factors is analogous to the function of the sigma proteins in bacteria, except that BTF interact with DNA independent of RNA polymerase − Research showed several important distinctions about how transcription works in bacteria and eukaryotes o In bacteria a single sigma protein binds to a promoter and initiates transcription, but in eukaryotes many BSL are required to initiate transcription  In eukaryotes the machinery required to start transcription is complex o Eukaryotes have three distinct types of RNA polymerase, instead of just one  RNA pol I transcribes genes that code for most of the large RNA molecules found in ribosomes  RNA pol II transcribes protein-coding genes (produces mRNAs)  RNA pol III transcribes genes that code for tRNA and genes that code for one of the small RNA molecules found in ribosomes  RNA pol II and pol III transcribe RNA molecules found in snRNPs o Although eukaryotic genomes contain promoters that signal where transcription should begin, just as bacteria do, the promoters in eukaryotic DNA are much more diverse and complex than bacterial promoters  Many of the eukaryotic promoters recognized by RNA polymerase II include a unique sequence called the TATA box, located 30 bp upstream of the transcription start site  Some of the promoters recognized by pol II do not contain a TATA box  In addition, RNA pol I and pol III interact with entirely different promoters o In eukaryotes, transcription is followed by several important RNA processing steps that result in production of an mRNA that leaves the nucleus − The Discovery of Eukaryotic Genes in Pieces o Phillip Sharp and Richard Roberts proposed that there is not a one-to-one correspondence between the nucleotide sequence of a eukaryotic gene and its mRNA o Sections of non-coding sequence must be removed from the mRNA before it can carry an intelligible message to the translation machinery o When it became clear that the genes-in-pieces hypothesis was correct, Walter Gilbert suggested that regions of eukaryotic genes that are part of the final mRNA be referred to as exons (expressed) and the untranslated stretches as introns (they are intervening) o Exons code for segments of functional proteins or RNAs, introns do not o Introns are sections of genes that are not represented in the final mRNA product o As a result, eukaryotic genes are much larger than their corresponding mature RNA transcripts − Exons, Introns and RNA Splicing o The transcription of eukaryotic genes by RNA polymerase generates a primary RNA transcript that contains both the exon and intron regions o As transcription proceeds, the introns are removed from the growing RNA strand by a process known as splicing o In this phase, pieces of primary transcript are removed and the remaining segments are joined together o Splicing occurs while transcription is still under way and results in an RNA that contains an uninterrupted genetic message o Splicing is catalyzed by a complex of proteins and small RNAs known as small nuclear ribonucleoproteins or snRNPs o The process begins when an snRNP binds to the 5’ exon-intron boundary o Once the initial snRNPs are in place, other snRNPs arrive to form a mulitpart complex called a spliceosome o Once the spliceosome forms, the intron forms a loop with an adenine ribonucleotide at its base o The adenine participates in a reaction that cuts the loop out o A phosphodiester bond then links the exons on either side, forming a contiguous coding sequence o Splicing is complete and in most cases, the excised intron is degraded to ribonucleotide monophosphates o Current data suggests that both the cutting and rejoining reactions that occur during splicing are catalyzed by RNA molecules in the spliceosome Adding Caps and Tails to Transcripts − As soon as the 5’ end of a eukaryotic RNA emerges from RNA polymerase, enzymes add a structure called the 5’ cap − Cap consists of the molecule 7-methylguanylate and three phosphate groups − Also, an enzyme cleaves the 3’ end of most RNAs once transcription is complete and another enzyme adds a long tract of 100-250 adenine nucleotides, the sequence called a poly(A) tail − These tails are not encoded on the template strand − With the addition of the cap and tail, processing of the primary RNA transcript is complete − The product is a mature RNA − Experimental mRNAs that have a cap and a tail last longer when they are introduced into cells than do experimental mRNAs that lack a cap, a tail or both − mRNAs with caps and tails also produce more proteins than do mRNAs without caps and tails − The 5’ cap serves as a recognition signal for the translation machinery and the poly(A) tail extends the life span of an mRNA by protecting the message from degradation by ribonucleases in the cytosol An Introduction to Translation − Ribosomes are the Site of Protein Synthesis o There is a strong correlation between the number of ribosomes in a given type of cell and the rate at which that cell synthesizes proteins o Researchers confirmed the ribosome hypothesis using the pulse-chase experiment where they fed a pulse of radioactive sulphate to growing cultures of E. coli  They expected the cells to incorporate the radioactive sulphur into the amino acids methionine and cysteine, which contains sulphur, and then into the newly synthesized proteins  Fifteen seconds after adding the radioactive sulphate, they added the chase, which was a large excess of non-radioactive sulphate to the culture medium  Soon after the pulse of labelled sulphate ended, radioactive atoms were found in free amino acids or in ribosomes  Later, all of the radioactive atoms were found on completed proteins o Further study confirmed that in bacteria, ribosomes attach to mRNAs and begin synthesizing proteins even before transcription is complete o Transcription and translation can occur concurrently in bacteria because there is no nuclear envelope to separate the two processes o In eukaryotes, RNAs are processed in the nucleus and then mRNAs are exported to the cytoplasm o Once mRNAs are outside the nucleus, ribosomes attach to them and begin translation − How an mRNA Triplet Specifies an Amino Acid o Crick proposed that some sort of adapter molecule holds amino acids in place while interacting directly and specifically with a codon in mRNA via hydrogen bonding The Role of Transfer RNA − Crick’s adapter molecule was discovered by accident − Biologists were trying to work out a cell-free protein synthesis system derived from mammalian liver cells and had discovered that ribosomes, mRNA, amino acids, ATP and GTP had to present for translation to occur − In addition, a cellular fraction that contained a previously unknown type of RNA turned out to be indispensable − If this type of RNA is missing, protein synthesis does not occur − This new RNA became known as transfer RNA (tRNA) − A tRNA molecule that becomes covalently linked to an amino acid is called an aminoacyl tRNA − More recent research has shown that enzymes called aminoacyl tRNA synthetases are responsible for catalyzing the addition of amino acids to tRNAs − How Amino Acids are Loaded onto tRNAs o Active site on aminoacyl tRNA synthetase binds ATP and amino acid  Each aminoacyl tRNA synthetase is specific to one amino acid o Reaction leaves AMP and amino acid bound to enzyme; two phosphate groups are released o Activated amino acid is transferred from tRNA synthetase to the tRNA specific to
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