Notes on Replication, Transcription and Translation By Allen Johnny Borlay (MSc) Instructor, BIOLOGY 305 Section 3 & 4 Transcription overview The ﬁrst stage in the expression of genetic information is transcription of the information in the base sequence of a ds DNA to form the base sequence of a ss RNA. For any particular gene, only one strand of the DNA molecule, called the template strand, is copied by RNA polymerase. Because RNA polymerase moves in the 3' to 5' direction along the template strand of DNA, the RNA product is antiparallel and complementary to the template. RNA polymerase recognizes start signals (promoters) and stop signals (terminators) for each of the thousands of transcription units in the genome of an organism. Involves three processes: Initiation Elongation Termination Initiation Locate the initiation point and unwind the DNA double helix A group of proteins called initiation factors move along the DNA until it reach the promoter region special region of nucleotides that signal RNA polymerase to join the protein complex and begin synthesis of the RNA strand The promoter region usually does not vary too much from organism to organism The most common sequence of DNA nucleotides found on the promoter region is called the consensus sequence Elongation Once RNA polymerase unwinds the DNA, it creates a bubble that exposes the singlestrands of the DNA Transcription bubble moves down DNA at constant rate leaving growing RNA strands protruding from the bubble Only one of these DNA strands (template or antisense strand) is actually used to synthesize the RNA molecule Hydrogen bonds are created between the bases, phosphodiester bonds between the ribonucleotides. The other stand that is not used is called sense or coding strand During transcription, the bubble is maintained within bacterial RNA polymerase, which unwinds and rewinds DNA, maintains the conditions of the partner and template DNA strands, and synthesizes RNA. Termination Here sequence of nucleotides terminate transcription and this sequence is known as the termination sequence. When RNA polymerase reaches this termination sequence, a special protein assist in the dissociation process Stop sequences at the end of the gene cause phosphodiester bond formation to cease, transcription bubble to dissociate, and RNA polymerase to release DNA The product is immature RNA or pre mRNA (Primary transcript) The primary product of RNA transcription; the hnRNAs contain both intronic and exonic sequences. These hnRNAs are processed in the nucleus to give mature mRNAs that are transported to the cytoplasm where to participate in protein synthesis Eukaryotic transcription differs from prokaryotic transcription: three RNA polymerase enzymes initiation complex forms at promoter RNAs are modiﬁed after transcription The prokaryotic RNA-pol can bind to the DNA template directly in the transcription process. The eukaryotic RNA-pol requires co-factors to bind to the DNA template together in the transcription process Eukaryotes Transcription Regulation very complex Three different pols Distinguished by -amanitin sensitivity Pol I—rRNA, least sensitive Pol II– mRNA, most sensitive Pol III– tRNA , moderately sensitive Each polymerase recognizes a distinct promoter RNA polymerase in Eukaryotes RNA polymerase I transcribes rRNA RNA polymerase II transcribes hnRNA RNA polymerase III transcribes tRNA and other small RNAs Eukaryotic Polymerase I Promoters RNA Polymerase I Transcribes rRNA Sequence not well conserved Two elements Core element- surrounds the transcription start site (-45 to + 20) Upstream control element- between -156 and -107 upstream Spacing affects strength of transcription Eukaryotic Polymerase II Promoters Much more complicated Two parts Core promoter Upstream element Core promoter TATA box at ~-10 bases Initiator—on the transcription start site Downstream element-further downstream Many natural promoters lack recognizable versions of one or more of these sequences RNA pol III Precursors to tRNAs,5SrRNA, other small RNAs Promoter Type I Lies completely within the transcribed region 5SrRNA promoter split into 3 parts tRNA promoters split into two parts Polymerase II-like promoters EX. snRNA Lack internal promoter Resembles pol II promoter in both sequence and position Post-transcriptional processing of mRNA The mRNA that id formed directly after transcription is known as the precursor mRNA/pre-mRNA. This pre-mRNA has not yet undergone the necessary modiﬁcation processes. In the nucleus, before the RNA can enter the cytoplasm, the pre-mRNA must undergo three post-transcriptional modiﬁcations. 1. 5’ Guanosine Triphosphate Cap 2.Polyadenylation of the 3’ end and 3. Splicing of Exons 5’ Guanosine Triphosphate Cap In the nucleus of the cell, the 5’ end of the pre-mRNA is altered by the attachment of a guanosine nucleotide via a 5’-5’ triphosphate linkage. The guanosine nucleotide is quickly methylated at the 7th position to form the 7-methyl guanosine Why attach the 5’-guanosine cap? One important of this cap is to protect the mRNA from degradation during protein synthesis. It also stabilizes the mRNA and aids in transport across the nuclear membrane. Polyadenylation of the 3’ end In this type of post-transcriptional modiﬁcation, the 3’-end of the pre-mRNA is removed and a series of adenosine nucleotides are added. Therefore, the 3’-end tail that contains the many adenines is called the polyadenine tail Just as the 5’cap adds stability and provides resistance to degradation, the poly-(A) tail also provide the mRNA with stability and keeps the tail from degradation Splicing of Exons Not all regions of the pre-mRNA molecule code for proteins Those regions that do code for proteins are called exons while those that do not code for proteins are called introns Before the pre-mRNA exits the nucleus , special proteins/enzymes will cut out the introns and splice together the exons TRANSLATION Conversion of a messenger RNA sequence into the amino acid sequence of a polypeptide (i.e., protein synthesis) Translation occurs in the cytoplasm where ribosomes are located mRNA carries the genetic code to cytoplasm, where it is translate by ribosomes. Ribosomal RNA forms an integral part of the ribosome, without which translation would not be possible. Transfer RNA collects the proper amino acids and bring them to the ribosomes where they are use to synthesize the polypeptide chain Three step involve: Initiation, Elongation and Termination Ribosomes Translation involves special cell machinery known as ribosomes. A given ribosome consist of two subunit, the small and large subunit These subunits consist of a combination of ribosomal RNA and proteins. Ribosomal RNA is synthesized in the nucleolus and travel to the cytoplasm, where they combine with the mRNA to form ribosomes Prokaryotes and Eukaryotes have ribosomes that are compose of slightly different subunits: Small subunit Large subunit Combine Eukaryotes 40S 60S 80S Prokaryotes 30S 50S 70S S stand for Svebery unit, which are the units that measure the rate at which a given particle or molecule sediments The svebery units usually refers to how something travel down a test tube when it is rotating with some angular velocity Ribosomes cont. S stand for Svebery unit, which are the units that measure the rate at which a given particle or molecule sediments The svebery units usually refers to how something travel down a test tube when it is rotating with some angular velocity The small and large subunits only come together during the process of translation. The purpose of ribosome is to use the genetic code to translate the sequence of nucleotide on the mRNA into correct sequence of amino acids, thereby synthesizing the protein Translation _ Initiation Once the pre-mRNA undergoes the proper modiﬁcation in the nucleus, it becomes mRNA. mRNA then travels through the nuclear pores and into the cytoplasm of the cell. Once inside the cytoplasm, with the help from proteins called initiation factors, the small subunit binds to 5’ end of the mRNA. The small subunit slides along with mRNA until it reaches the nucleotide sequence 5’—AUG—3’. This the start codon and it signals for tRNA to bring the amino acid methionine Once the tRNA with the methionine binds to the start codon, the large subunit binds to the small subunit thereby completing the ribosome. During initiation, the mRNA locate the small ribosomal subunit and bind to it with the help of protein called initiation factors. The small subunit move along the mRNA until it reaches the start codon. Translation _ Initiation cont. This signals a transfer RNA molecular to ﬁnd the methionine amino acid attach to it forming the aminoacyl transfer RNA complex This complex then binds to the start codon which signals the large ribosomal subunit to combine with the small subunit, thereby forming the ribosome The structure of tRNA is especially suitable for it function- towards the middle of the RNA strand is a sequence of nucleotides that is complementary to the start codon sequence; this is known as anticodon of the tRNA The 3’ end of the tRNA contains nucleotide sequence that binds the appropriate amino acids. An enzyme called tRNA synthetase catalyzes the binding of the tRNA to appropriate amino acid Translation _ Elongation Once the ribosome complex has been formed and the tRNA carries the methionine is in the P-site, the ribosome can now begin sliding along the mRNA The next aminoacyl tRNA that carries the next amino acid is brought into the Asite. After the P and A sites are ﬁlled, an enzyme called peptidyl transferase catalyzes the formation of peptide bond between the amino acids in the P-site and the A-site. When the peptide bod is formed, the amino acid detaches from tRNA in the P-site The ribosome complex moves three nucleotides in the 5’ to 3’ direction along the mRNA. This move the tRNA in the P-site into E-site and it relocate the tRNA carrying the growing polypeptide into the P-site Once the tRNA moves into the E-site, it is then expelled out of the ribosome complex. This sliding process is known as translocation After the A-site is empty, the process can repeat itself to add another amino acid. This continues until the polypeptide chain is complete Translation _ Termination Just like there is a start codon that signals initiation, there is a stop codon that signals termination If the ribosome reads either UAA, UGA or UAG, a protein called release factor will bind to A-site This will ultimately cause the polypeptide chain to break off of the tRNA in the P-site This then give rise to the ribosome complex to dissociate and this will end translation. Post-translation activities Once the polypeptide is synthesized, it is usually modiﬁed before it can be a mature and active protein. These modiﬁcations are called post-translation. Six types of these modiﬁcation include 1. Phosphorylation Certain amino acids, such as serine, threonine and tyrosine found on the polypeptide chain can be modiﬁed via phosphorylation by enzymes called protein tinases. This type of modiﬁcation plays a crucial role in the cell cycle and signal transduction 2. Methylation Methyl groups (CH3) can be added onto the amino acid by an enzyme called methyltransferase. Methylation usually increases the hydrophobic character of the amino acid. Post-translation activities 3. Glycosylation This is one of the way in which polypeptides are modiﬁed. This process involves addition sugar components to the proteins. This can affect the proteins conformation and folding. One example of proteins that are glycosylated are membrane proteins that act as receptors for important biological molecules. 4. Proteolysis Certain proteins are synthesized in their inactive (zymogen) form. In order to activate them, enzymes called proteases must break certain peptide bonds. Many of the digestive enzymes in the small intestine use this type of posttranslation modiﬁcation Post-translation activities cont. 5. N-Acetylation N-acetylation is the transfer of an acetyl group onto the nitrogen on amino acid. This process can take place when the ribosome is still translating the polypeptide chain. In most eukaryotic cells, when translation is still taking place, the ﬁrst amino acid (met) is usually removed and replaced by an acetyl group. N-acetylation plays a crucial role in gene expression. Histones, the protein that assist in condensing DNA into chromatins can be acetylated, which reduces their ability to fold and open up the DNA for transcription 6. Lipidation Lipidation is the process by which lipid components are added onto the polypeptides. Usually those proteins that are destined to be in membranes, such as ER, mitochondria or plasma membranes undergo this process. It increases the proteins afﬁnity to the membranes.