Molecular Biology Lecture Notes PDF

Molecular Biology Dr Nzar Shwan MLT department Lecture 04: Monday, 14th October 2024 4th Year (Semester 7) Transcription: from DNA to RNA Central Dogma of molecular biology: Specifies that t...

Molecular Biology Dr Nzar Shwan MLT department Lecture 04: Monday, 14th October 2024 4th Year (Semester 7) Transcription: from DNA to RNA Central Dogma of molecular biology: Specifies that the usual flow of genetic information is from DNA to mRNA to polypeptide. It forms a cornerstone of our understanding of genetics at the molecular level. DNA is transcribed (copied) to mRNA which is translated to proteins using amino acids. During expression of a protein-coding gene, information flows from DNA → RNA → protein. This directional flow of information is known as the central dogma of molecular biology (Figure 1). Figure 1: The central dogma of molecular biology. The usual flow of genetic information is from DNA to mRNA to polypeptide. Note: The direction of informational flow shown in this figure is the most common direction, but exceptions occur. For example, RNA viruses and certain transposable elements use an enzyme called reverse transcriptase to make a copy of DNA from RNA. 1 Transcription is the mechanism by which a template strand of DNA is used by specific RNA polymerases to generate one of the three different types of RNA [Messenger RNA (mRNA)]. Types of RNA 1. Messenger RNA (mRNA) This class of RNAs are the genetic coding templates used by the translational machinery to determine the order of amino acids incorporated into an elongating polypeptide in the process of translation. 2. Transfer RNA (tRNA) This class of small RNAs form covalent attachments to individual amino acids and recognize the encoded sequences of the mRNAs to allow correct insertion of amino acids into the elongating polypeptide chain. 3. Ribosomal RNA (rRNA) This class of RNAs are assembled, together with numerous ribosomal proteins, to form the ribosomes. Where does transcription take place? Overview of transcription Transcription is the first step in gene expression, in which information from a gene is used to construct a functional product such as a protein. The goal of transcription is to make an RNA copy of a gene's DNA sequence. Transcription is performed by enzymes called RNA polymerases, which link nucleotides to form an RNA strand (using a DNA strand as a template). For a protein-coding gene, the RNA copy, or transcript, carries the information needed to build a polypeptide (protein or protein subunit). Transcription has three stages: initiation, elongation, and termination. 2  In eukaryotes, RNA molecules must be processed after transcription (post transcriptional modifications): they are spliced and have a 5' cap and poly-A tail put on their ends. Transcription is controlled separately for each gene in your genome. RNA polymerase The main enzyme involved in transcription is RNA polymerase, which uses a single-stranded DNA template to synthesize a complementary strand of RNA. 3 Specifically, RNA polymerase builds an RNA strand in the 5' to 3' direction, adding each new nucleotide to the 3' end of the strand. RNA polymerases are large enzymes with multiple subunits, even in simple organisms like bacteria. Humans and other eukaryotes have three different kinds of RNA polymerase: I, II, and III. Each one specializes in transcribing certain classes of genes. Stages of transcription Transcription of a gene takes place in three stages: initiation, elongation, and termination. Here, we will briefly see how these steps happen in bacteria (Prokaryotes). 1. Initiation RNA polymerase binds to a sequence of DNA called the promoter, found near the beginning of a gene. Each gene (or group of co-transcribed genes, in bacteria) has its own promoter. The promoter sequence directs the exact location for the initiation of RNA transcription. Most of the promoter region is located just ahead of or upstream from the site where transcription of a gene begins. Once bound, RNA polymerase separates the DNA strands, providing the single-stranded template needed for transcription. 4 2. Elongation After the initiation stage of transcription is completed, the RNA transcript is made during the elongation stage (Figure 2). During the synthesis of the RNA transcript, RNA polymerase moves along the DNA, causing it to unwind. One strand of DNA, the template strand, acts as a template for RNA polymerase. The opposite DNA strand is the coding strand (also called the sense strand). As it "reads" this template one base at a time, the polymerase builds an RNA molecule out of complementary nucleotides, making a chain that grows from 5' to 3'. 5  The RNA transcript carries the same information as the non- template (coding) strand of DNA, but it contains the base uracil (U) instead of thymine (T). [What do 5' and 3' mean?] Figure 2: Synthesis of the RNA transcript. Transcription of multiple genes When considering the transcription of multiple genes within a chromosome, the direction of transcription and the DNA strand used as a template varies among different genes. Figure 3 shows three genes adjacent to each other within a chromosome. Genes A and B are transcribed from left to right, using the bottom DNA strand as a template. By comparison, gene C is transcribed from right to left and uses the top DNA strand as a template. Note that in all three cases, the template strand is read in the 3ʹ to 5ʹ direction, and the synthesis of the RNA transcript occurs in a 5ʹ to 3ʹ direction. 6  Figure 3 : The transcription of three different genes found in the same chromosome. RNA polymerase synthesizes each RNA transcript in a 5ʹ to 3ʹ direction, sliding along a DNA template strand in a 3ʹ to 5ʹ direction. However, the use of the template strand varies from gene to gene. For example, genes A and B use the bottom strand, but gene C uses the top strand. 3. Termination Sequences called terminators signal that the RNA transcript is complete. Once they are transcribed, they cause the transcript to be released from the RNA polymerase. An example of a termination mechanism involving formation of a hairpin in the RNA is shown below. 7 Transcription in eukaryotes Key points: Promoter structure In eukaryotes, the promoter sequence is more variable and often more complex than that found in bacteria. For structural genes, at least three features are found in most promoters: regulatory elements, a TATA box, and a transcriptional start site. The core promoter is a relatively short DNA sequence that is necessary for transcription to take place. It consists of a TATAAA sequence called the TATA box and the transcriptional start site, where transcription begins. The TATA box, which is usually about 25 bp upstream from a transcriptional start site, is important in determining the precise starting point for transcription. If it is missing from the core promoter, the transcription start site becomes undefined, and transcription may start at a variety of different locations. Regulatory elements are short DNA sequences that affect the ability of RNA polymerase to recognize the core promoter and begin the process of transcription. o Enhancers are needed to stimulate transcription. o Silencers—DNA sequences that inhibit transcription. Pre- mature and mature mRNA transcripts When an RNA transcript is first made in a eukaryotic cell, it is considered a pre-mRNA and must be processed into a messenger RNA (mRNA). A 5' cap is added to the beginning of the RNA transcript, and a 3' poly-A tail is added to the end. In splicing, some sections of the RNA transcript (introns) are removed, and the remaining sections (exons) are stuck back together. Some genes can be alternatively spliced, leading to the production of different mature mRNA molecules from the same initial transcript. 8 Eukaryotic RNA modifications In bacteria, RNA transcripts can act as messenger RNAs (mRNAs) right away. In eukaryotes, the transcript of a protein-coding gene is called a pre-mRNA and must go through extra processing before it can direct translation.  Eukaryotic pre-mRNAs must have their ends modified, by addition of a 5' cap (at the beginning) and 3' poly-A tail (at the end). o 5' cap: The cap is a modified guanine (G) nucleotide, which:  Protects the transcript from being broken down.  Helps the ribosome attach to the mRNA and start reading it to make a protein. o 3' poly-A tail: When a sequence called a polyadenylation signal shows up in the RNA molecule during transcription, an enzyme cuts the RNA in two at that site. Another enzyme adds about 100 - 200 adenine (A) nucleotides to the cut end, forming a poly-A tail.  The tail makes the transcript more stable  It helps the transcript get exported from the nucleus to the cytosol.  Many eukaryotic pre-mRNAs undergo splicing. In this process, parts of the pre-mRNA (called introns) are chopped out, and the remaining pieces (called exons) are stuck back together. End modifications increase the stability of the mRNA, while splicing gives the mRNA its correct sequence. 9