7. Transcription - from DNA to RNA.pdf

Molecular Biology and Cytogenetics 7. Transcription - from DNA to RNA Instructor: Dr. Mohammad Abukhalil RNA (ribonucleic acid) ⚫ RNA is a linear polymer made of four different types of nucleotide subunits linked together by phosphodiester bonds. ⚫ It differs...

Molecular Biology and Cytogenetics 7. Transcription - from DNA to RNA Instructor: Dr. Mohammad Abukhalil RNA (ribonucleic acid) ⚫ RNA is a linear polymer made of four different types of nucleotide subunits linked together by phosphodiester bonds. ⚫ It differs from DNA chemically in two respects: (1) The nucleotides in RNA are ribonucleotides—that is, they contain the sugar ribose (hence the name ribonucleic acid) rather than deoxyribose. (2) Although, like DNA, RNA contains the bases adenine (A), guanine (G), and cytosine (C), it contains the base uracil (U) instead of the thymine (T) in DNA. Six major types of RNA ⚫ Ribosomal RNA (rRNA) ⚫ Messenger RNA (mRNA) ⚫ Transfer RNA (tRNA) ⚫ Small nuclear RNA (snRNA) ⚫ Small nucleolar RNA (snoRNA) ⚫ MicroRNA (miRNA) Two key points for understanding RNA function ⚫ RNA can form complementary base pairs with other nucleic acids. ⚫ RNA can interact with proteins: Ribonucleoprotein (RNP) particles Ribosomal RNA (rRNA) ⚫ rRNA accounts for approximately 80% of total RNA in the cell. ⚫ rRNA is an important structural and functional part of the ribosomes (cellular complex molecule where proteins are synthesized). ⚫ Ribosomes are important during protein synthesis as they contain peptidyl transferase “activity,” an activity catalyzed by ribozymes that participate in peptide bond formation. Transfer RNA (tRNA) ⚫ tRNA is the smallest of the three RNAs. ⚫ tRNA is helps decode a mRNA sequence into a protein. Messenger RNA (mRNA) ⚫ mRNA carries genetic information from DNA to cytosol for translation. ⚫ About 5% of the total RNA within a cell is mRNA and is a single strand of nucleotides known as ribonucleic acid. ⚫ During the transcription process, a single strand of DNA is decoded by RNA polymerase, and mRNA is synthesized. Overview of the versatility of RNA ⚫ RNA can serve as a “scaffold” upon which proteins can be assembled. e.g. signal recognition particle ⚫ RNA-protein interactions can influence the catalytic activity of proteins. e.g. telomerase ⚫ RNA can be catalytic. ⚫ Small RNAs can directly control gene expression. ⚫ RNA can be the hereditary material. Gene expression  Gene expression ⚫ The DNA inherited by an organism leads to specific traits by dictating the synthesis of RNA molecules involved in protein synthesis. ⚫ Gene expression is the process by which DNA directs the synthesis of proteins. ⚫ The expression of genes that code for proteins includes two stages: transcription and translation. ⚫ Transcription is the synthesis of RNA using information in the DNA. The gene ⚫ A gene is a sequence of genomic nucleic acids that codes for a molecule that has a function. ⚫ Eukaryotic genes are composed of coding exons, noncoding introns, and noncoding consensus sequences. ⚫ Intron’s and exon’s number, size, location, and sequence differ from gene to gene. Different genes are transcribed at different levels, meaning that different numbers of RNA molecules are made for each. Prokaryotic vs Eukaryotic gene expression ⚫ Eukaryotic gene expression occurs in both the nucleus (transcription) and cytoplasm (translation); in prokaryotes, gene expression (both transcription and translation) occurs within the cytoplasm of a cell due to the lack of a defined nucleus. ⚫ Eukaryotes have monocistronic genes i.e. one messenger RNA molecule can encode for only one polypeptide; whereas, prokaryotes have polycistronic genes meaning one messenger RNA molecule can encode more than one polypeptide or one mRNA can produce two or more proteins. ⚫ Eukaryotic genes are split into exons and introns; in prokaryotes, genes are almost never split. ⚫ In eukaryotes, mRNA is synthesized in the nucleus and then processed and exported to the cytoplasm; in bacteria, transcription and translation can take place simultaneously off the same piece of DNA. ⚫ Much of eukaryotic DNA does not code for proteins (~98% is non-coding in humans); in bacteria often more than 95% of the genome codes for proteins. Molecular components of transcription ⚫ RNA polymerase ⚫ The promoter ⚫ Transcription unit ⚫ Termination sequences Transcription Enzymes ⚫ RNA polymerase: The enzyme that controls transcription. ⚫ RNA polymerases can assemble a polynucleotide only in its 5' to 3' direction. ⚫ Unlike DNA polymerases, RNA polymerases are able to start a chain from scratch; they don’t need a primer.  RNA polymerases ⚫ There are several distinct RNA polymerases in eukaryotic cells. ⚫ RNA polymerase I synthesizes rRNAs involved in facilitating protein synthesis by the ribosome. ⚫ RNA polymerase II is responsible for the synthesis of mRNA and miRNAs. ⚫ RNA polymerase III catalyzes the synthesis of tRNAs.  Eukaryotic Promoter ⚫ Promoter is the DNA sequence where RNA polymerase attaches and initiates transcription. ⚫ The consensus sequence for promoters typically has the sequence “TATA” (or variations of T and A) and is often located 15 to 30 base pairs (bp) upstream from the transcription start site, called a TATA box. ⚫ Additional sequences that may be required for promoter function include the CAAT box and the GC box. Transcription unit ⚫ A transcription unit is a linear sequence of DNA that extends from a transcription start site to a transcription stop site. Stages of transcription ⚫ Initiation ⚫ Elongation ⚫ Termination 1. Initiation ⚫ Initiation of transcription occurs at the transcription start point (the nucleotide where RNA synthesis actually begins) in the promoter. ⚫ In eukaryotes, a collection of proteins called transcription factors mediate the binding of RNA polymerase and the initiation of transcription. Only after transcription factors are attached to the promoter does RNA polymerase II bind to it. ⚫ Several transcription factors, one recognizing the TATA box in the promoter, must bind to the DNA before RNA polymerase II can bind in the correct position and orientation. ⚫ The whole complex of transcription factors and RNA polymerase II bound to the promoter is called a transcription initiation complex.  Initiation ⚫ Once the appropriate transcription factors are firmly attached to the promoter DNA and the polymerase is bound in the correct orientation, the enzyme unwinds the two DNA strands and begins transcribing the template strand at the start point. ⚫ In eukaryotes, in RNA polymerase II-dependent transcription, there are six general transcription factors: TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. 2. Elongation ⚫ RNA polymerase creates a transcription bubble, which separates the two strands of the DNA helix. This is done by breaking the hydrogen bonds between complementary DNA nucleotides. ⚫ One strand of the DNA, the template strand (or noncoding strand), is used as a template for RNA synthesis. ⚫ RNA polymerase reads DNA 3′-5′ and produces only in its 5' to 3' direction. ⚫ A single gene can be transcribed simultaneously by several molecules of RNA polymerase following each other which helps the cell make the encoded protein in large amounts. Elongation ⚫ In the wake of this advancing wave of RNA synthesis, the new RNA molecule peels away from its DNA template, and the DNA double helix re- forms. ⚫ Transcription progresses at a rate of about 40 nucleotides per second in eukaryotes. ⚫ The coding (non-template) strand and newly formed RNA can also be used as reference points, so transcription can be described as occurring 5' → 3'. This produces an RNA molecule from 5' → 3', an exact copy of the coding strand (except that thymines are replaced with uracils, and the nucleotides are composed of a ribose (5-carbon) sugar where DNA has deoxyribose (one fewer oxygen atom) in its sugar-phosphate backbone) Termination of Transcription ⚫ The mechanism of termination differs between bacteria and eukaryotes. ⚫ In bacteria, transcription proceeds through a terminator sequence in the DNA. ⚫ The transcribed terminator (an RNA sequence) functions as the termination signal, causing the polymerase to detach from the DNA and release the transcript, which requires no further modification before translation. Termination of Transcription ⚫ In eukaryotes, RNA polymerase II transcribes a sequence on the DNA called the polyadenylation signal sequence, which specifies a polyadenylation signal (AAUAAA) in the pre-mRNA. ⚫ This is called a “signal” because once this stretch of six RNA nucleotides appears, it is immediately bound by certain proteins in the nucleus. ⚫ Then, at a point about 10–35 nucleotides downstream from the AAUAAA, these proteins cut it free from the polymerase, releasing the pre-mRNA. The pre-mRNA then undergoes processing. Termination of Transcription ⚫ The transcript is cleaved at an internal site before RNA Polymerase II finishes transcribing. ⚫ The remainder of the transcript is digested by a 5′-exonuclease (called Xrn2 in humans) while it is still being transcribed by the RNA Polymerase II. When the 5′-exonulease “catches up” to RNA Polymerase II by digesting away all the overhanging RNA, it helps disengage the polymerase from its DNA template strand, finally terminating that round of transcription. Transcriptional enhancers ⚫ In some eukaryotic genes, some promoters work in concert with other types of regulatory sequences known as enhancers, which sometimes lie upstream of a gene, within the coding region of the gene, downstream of a gene, or may be thousands of nucleotides away. ⚫ Enhancer regions serve as binding sites for proteins known as activators that help increase or enhance transcription. RNA processing reactions ⚫ Gene transcription produces an RNA that is larger than the mRNA found in the cytoplasm for translation. ⚫ This larger RNA, called the primary transcript, contains segments of transcribed introns. For example, a precursor mRNA (pre-mRNA) is a type of primary transcript that becomes a messenger RNA (mRNA) after processing. ⚫ The intron segments are removed and the exons are joined at specific sites, called donor and acceptor sequences, to form the mature mRNA by a mechanism of RNA processing. I- Addition of a 5′ cap ⚫ Almost immediately after the initiation of RNA synthesis, the 5′ end of RNA is capped by a methyl guanine (G) nucleotide added onto the 5' end after transcription of the first 20–40 nucleotides.  II- Addition of a poly(A) tail ⚫ The 3' end of the pre-mRNA molecule is also modified before the mRNA exits the nucleus. ⚫ Recall that the pre-mRNA is released soon after the polyadenylation signal, AAUAAA, is transcribed. ⚫ At the 3' end, an enzyme then adds 50–250 more adenine (A) nucleotides, forming a poly-A tail. The 5' cap and poly-A tail share several important functions ⚫ They protect RNA from degradation (by 5′ exonucleases) during elongation of the RNA chain. ⚫ They seem to facilitate the export of the mature mRNA from the nucleus. ⚫ They help ribosomes attach to the 5' end of the mRNA once the mRNA reaches the cytoplasm.  RNA Splicing ⚫ A remarkable stage of RNA processing in the eukaryotic nucleus is the removal of large portions of the RNA molecule that is initially synthesized. ⚫ This cut-and-paste job, called RNA splicing, is a form of RNA processing in which a newly made precursor messenger RNA (pre- mRNA) transcript is transformed into a mature messenger RNA (mRNA). ⚫ The average length of a transcription unit along a human DNA molecule is about 27,000 nucleotide pairs, so the primary RNA transcript is also that long. However, the average-sized protein of 400 amino acids requires only 1,200 nucleotides in RNA to code for it. Introns and exons ⚫ The noncoding segments of nucleic acid that lie between coding regions are called intervening sequences, or introns. The other regions are called exons, because they are eventually expressed, usually by being translated into amino acid sequences. ⚫ In making a primary transcript from a gene, RNA polymerase II transcribes both introns and exons from the DNA, but the mRNA molecule that enters the cytoplasm is an abridged version.  How is pre-mRNA splicing carried out? ⚫ Splice site sequences, which indicate the beginning (GU) and ending (AG) of each intron, are found within the primary RNA transcript. ⚫ The removal of introns is accomplished by a large complex made of proteins and small RNAs called a spliceosome. This complex binds to several short nucleotide sequences along an intron, including key sequences at each end. ⚫ The intron is then released (and rapidly degraded), and the spliceosome joins together the two exons that flanked the intron. ⚫ The mature RNA molecule now leaves the nucleus by passing into the cytoplasm through the pores in the nuclear membrane. Alternative splicing ⚫ The final protein products encoded by any given intron- exon sequence also vary in structure, depending on which exons are spliced back together during RNA processing. ⚫ This so-called "alternative splicing“ is a process by which different combinations of exons are joined together and result in the production of multiple forms of mRNA from a single pre-mRNA 58