Molecular Biology Wk 10 Flashcards
An Overview of the Flow of Information through the Cell
The central dogma of molecular biology is an explanation of the flow of genetic information within a
biological system.
RNA-based enzymatic catalysis is an essential part of the machinery for synthesizing proteins. While DNA duplication is driven by protein enzymes in modern organisms, the ability of DNA and RNA to base pair with free nucleotides suggests a scenario in which an RNA sequence spontaneously formed that was then able to self-replicate, ultimately giving rise to many self-propagating copies of itself. This is known as the “ RNA world ” hypothesis, which proposes that life began as RNA.
The Central Dogma
The first meaningful insight into gene function was gained by Archibald Garrod, a physician who reported in
1908 that the symptoms exhibited by persons with certain rare inherited diseases were caused by the absence of specific enzymes. Garrod had discovered the relationship between a genetic defect, a specific enzyme, and a specific metabolic condition. He called such diseases “inborn errors of metabolism.” One of the diseases investigated by Garrod was alcaptonuria , a condition readily diagnosed because the urine becomes dark on exposure to air. Garrod found that persons with alcaptonuria lacked an enzyme in their blood that oxidized homogentisic acid, a compound formed during the breakdown of the amino acids phenylalanine and tyrosine. As homogentisic acid accumulates, it is excreted in the urine and darkens in color when oxidized by air.
An overview of the flow of information in a eukaryotic cell.
The DNA of the chromosomes located within the nucleus contains the entire store of genetic information. Selected sites on the DNA are transcribed into pre-mRNAs (step 1), which are processed into messenger RNAs (step 2). The messenger RNAs are transported out of the nucleus (step 3) into the cytoplasm, where they are translated into polypeptides by ribosomes that move along the mRNA (step 4). Following translation, the polypeptide folds to assume its native conformation (step 5).
An important discovery
Retro viruses (e.g. HIV - human immunodeficiency virus) carry RNA as their genetic information. When they invade their host cell they convert their RNA into a DNA copy using reverse transcriptase.
Thus the central dogma is modified:
DNA to RNA to protein
Gene expression
Protein synthesis four steps:
Transcription
RNA processing
Translation
Post-translation processing
The Role of RNA Polymerases in Transcription
Transcription is a process in which a DNA strand provides the information for the synthesis of an RNA strand. The enzymes responsible for transcription in both prokaryotic and eukaryotic cells are called DNA-dependent RNA polymerases , or simply RNA polymerases. These enzymes are able to incorporate nucleotides, one at a time, into a strand of RNA whose sequence is complementary to one of the DNA strands, which serves as the template. RNA polymerase catalyzes the highly favorable reaction in which ribonucleoside triphosphate substrates (NTPs) are cleaved into nucleoside monophosphates as they are polymerized into a covalent chain.
The Role of RNA
Polymerases in Transcription /cont./
The first step in the synthesis of an RNA is the association of the polymerase with the DNA template. The site on the DNA to which an RNA polymerase molecule binds prior to initiating transcription is called the promoter .
Cellular RNA polymerases are not capable of recognizing promoters on their own but require the help of additional proteins called transcription factors .
In addition to providing a binding site for the polymerase, the promoter contains the information that determines which of the two DNA strands is transcribed and the site at which transcription begins. RNA polymerase moves along the template DNA strand toward its 5 ′ end (i.e., in a 3 ′ → 5 ′ direction). As the polymerase progresses, the DNA is temporarily unwound, and the polymerase assembles a complementary strand of RNA that grows starting from its 5 ′ terminus in a 3 ′ direction
(FIGURE).
A schematic model of the elongation of a newly synthesized RNA molecule during transcription.
The polymerase covers approximately 35 base pairs of DNA, the transcription bubble composed of single-stranded (melted) DNA contains about 15 base pairs, and the segment present in a DNA–RNA hybrid includes about nine base pairs.
A transcription bubble is a molecular structure formed during DNA transcription when a limited portion of the DNA double strand is unwound.
The Role of RNA Polymerases in Transcription /cont./
LOOK AT GOODNOTES
RNA
Three major types of eukaryotic RNAs—mRNAs, rRNAs, and tRNAs—are derived from precursor RNA molecules that are considerably longer than the final RNA product. The initial precursor RNA is equivalent in length to the full length of the DNA transcribed and is called the primary transcript , or pre-RNA . The corresponding segment of DNA from which a primary transcript is transcribed is called a transcription unit . Primary transcripts do not exist within the cell as naked RNA but become associated with proteins even as they are synthesized. Primary transcripts typically have a fleeting existence, being processed into smaller, functional RNAs by a series of “cut-andpaste” reactions. RNA processing requires a variety of small RNAs (90 to 300 nucleotides long) and their associated proteins.
➢Messenger RNA (mRNA) <5%- mRNA provides the plan for the polypeptide chain;
➢Ribosomal RNA (rRNA) Up to 80%-rRNA provides the platform for protein synthesis;
➢Transfer RNA (tRNA) About 15%-tRNA “translates” the message on the mRNA into a polypeptide chain. ➢In eukaryotes small nuclear ribonucleoproteins (snRNP).
Transcription: The synthesis of a strand of mRNA (and other RNAs)
Uses an enzyme RNA polymerase
●Proceeds in the same direction as replication (5’
to 3’)
●Forms a complementary strand of mRNA
●It begins at a promotor site which signals the beginning of gene is not much further down the molecule (about 20 to 30 nucleotides)
●After the end of the gene is reached there is a terminator sequence that tells RNA polymerase to stop transcribing.
Transcription Enzyme - RNA polymerase:
The enzyme that controls transcription and is
characterized by:
➢Search DNA for initiation site,
➢It unwinds a short stretch of double helical DNA to produce a single-stranded DNA template,
➢It selects the correct ribonucleotide and catalyzes the formation of a phosphodiester bond,
➢It detects termination signals where transcript ends.
Transcription into 3 phases
●In eukaryotes, unformylated methionine is the initial amino acid of polypeptide synthesis.
●AUG, codes for methionine; Rarely, another codon, GUG, specifies methionine during initiation, though it is not clear why this happens, since GUG normally encodes valine.
●Three codons (UAG, UAA, and UGA) serve as termination codons.
Transcription in Bacteria
Bacteria, such as E. coli , contain a single type of RNA
polymerase composed of five subunits that are tightly associated to form a core enzyme .
In the absence of the σ factor, the core enzyme does not
interact with the DNA at specific initiation sites. When the core enzyme is associated with the σ factor, the complete enzyme (or holoenzyme) is able to recognize and bind to the promoter regions of the DNA, separate the strands of the DNA double helix, and initiate transcription at the proper start sites. In the traditional model shown here, the σ factor dissociates from the core enzyme, which is capable of transcription elongation. Several studies suggest that, in at least some cases, σ may remain with the polymerase. Bacterial promoters are located in the region of a DNA strand just preceding the initiation site of RNA synthesis. The nucleotide at which transcription is initiated is denoted as +1 and the preceding nucleotide as −1. Those portions of the DNA preceding the initiation site (toward the 3 ′ end of the template strand) are said to be upstream from that site. Those portions of the DNA succeeding it (toward the 5 ′ end of the template strand) are said to be downstream from that site.
As the σ factor interacts with the promoter, the jaws of the enzyme grip the downstream DNA duplex, which resides within the channel. The enzyme then separates (or melts ) the two DNA strands in the region surrounding the start site. The complex of the polymerase, σ factor, and DNA with the strands separated is called the Open Complex. Once about 10 nucleotides have been successfully incorporated into a growing transcript, the enzyme undergoes a major change in conformation and is transformed into a transcriptional elongation complex that can move processively along the DNA. In the model shown in Figure, the formation of an elongation complex is followed by release of the σ factor.
Transcription in Bacteria /cont./
Analysis of the bacterial RNA polymerase (see Figure) reveals a molecule shaped like a crab claw
with a pair of mobile pincers (or jaws) enclosing a positively charged internal channel.
RNA polymerases from the three domains of life.
Each subunit of an enzyme is denoted by a different color and labeled according to conventional nomenclature
for that enzyme. Homologous subunits are depicted by the same color.
Transcription in Bacteria /cont./
As noted earlier, promoters are the sites in DNA that bind RNA polymerase. Bacterial promoters are located in the region of a DNA strand just preceding the initiation site of RNA synthesis. Analysis of the DNA sequences just upstream from a large number of bacterial genes reveals that two short stretches of DNA are similar from one gene to another. One of these stretches is centered at approximately 35 bases upstream from the initiation site and typically occurs as the sequence TTGACA (Figure). This TTGACA sequence (known as the −35 element) is called a consensus sequence , which indicates that it is the most common version of a conserved sequence, but that some variation occurs from one gene to another. The second conserved sequence is found approximately 10 bases upstream from the initiation site and occurs at the consensus sequence TATAAT (Figure). This site in the promoter, named the – 10 element after its position or the Pribnow box after its discoverer, is responsible for identifying the precise nucleotide at which transcription begins. As the sigma factor recognizes the Pribnow box, amino acid residues within the protein interact with each of the six nucleotides of the TATAAT sequence of the nontemplate strand. Two of these nucleotides are flipped out of the nucleotide stack and into the core of the protein. This action likely initiates melting of the adjoining region of promoter DNA and formation of the transcription bubble.
The basic elements of a promoter region in the DNA of the bacterium E. coli.
The key regulatory sequences required for initiation of transcription are found in regions located at −35 and −10 base pairs from the site at which transcription is initiated. The initiation site marks the boundary between the + and − sides of the gene.
Transcription in Eukaryotes Differs from Prokaryotic
Transcription in Several Ways
●Transcription in eukaryotes occurs within the nucleus
●Requirement in eukaryotes for a large variety of accessory proteins, or transcription factors. These proteins play a role in virtually every aspect of the transcription process, from the binding of the polymerase to the DNA template, to the initiation of transcription, to its elongation and termination.
●Initiation of transcription of eukaryotic genes requires the compact chromatin fiber, characterized by nucleosome coiling, to be uncoiled and the DNA to be made accessible to RNA polymerase and other regulatory proteins.
●Initiation and regulation of transcription entail a more extensive interaction between cis-acting DNA sequences
and trans-acting protein factors involved in stimulating and initiating transcription. Eukaryotic RNA polymerases, for example, rely on transcription factors (TFs) to scan and bind to DNA.
●Alteration of the primary RNA transcript to produce mature eukaryotic mRNA involves many complex stages referred to generally as “processing.”
Eukaryotic gene structure
Most eukaryotic genes in contrast to typical bacterial genes, the coding sequences (exons) are interrupted by noncoding DNA (introns). The gene must have (Exon; start signals; stop signals; regulatory control elements).
