lecture5_TRANSCRIPTION AND RNA DIVERSITY (2015)Apunte Inglés
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Molecular biology – lecture 5
TRANSCRIPTION AND RNA DIVERSITY
The central dogma of molecular biology states that
information flows in the DNA RNA protein sense. All
sells from bacteria to humans express their genetic
information in this way. There are some viruses
(retroviruses) that don’t follow this rule and that
information flows in RNA DNA sense. This process only
belongs to RNA viruses and has been the key to fight against
retroviruses such as AIDS. In some other cases, DNA can be
copied into RNA in a process known as auto replication.
As two important differences to DNA, RNA has: - Uracil instead of thymine (this allows repairing of deamination cytosine to uracil in DNA) Ribose instead of deoxyribose, which makes RNA more unstable RNA is usually unstable, that allows to reset gene expression when we don’t need them.
2. TRANSCRIPTION Transcription is the process in which you copy one of the strands of DNA into RNA. This process has become similarities to the process of NDA replication. Transcription firstly starts with the opening and unwinding of a small portion of the DNA double helix. Then, only one strand will be copied, that will act as a template and that’s why we call it the template strand.
The RNA chain is determined by the complementary base-paring of the template strand. The other strand of DNA will be called the coding strand because it has the code of the new RNA chain; it’s equivalent to the RNA chain but instead of uracil it has thymine The enzymes that performed transcription are called RNA polymerases and as the DNA polymerases, they catalyze the formation of phosphodiester bonds that link the nucleotides together to form a linear chain. All polymerases only add nucleotides on the 3’ end and this rule is the same for RNA polymerases. The new chain is extended by one nucleotide by one Molecular biology – lecture 5 nucleotide at a time in the 5’3’ direction such as in DNA and it will be complementary to the template strand. Because new and template strands have to be antiparallel, depending on the direction of the DNA in the gene, the complementary strand will be different so the definition of coding strand and template strand will vary depending on the direction in which the gene is transcribed.
RNA polymerases don’t need a primer to start the synthesis of the new RNA chain because they do not check the last nucleotide added. Transcription doesn’t need to be as accurate as DNA replication; RNA polymerases make about one mistake every 104 nucleotides copied into RNA and the consequences of an error in RNA transcription are much less significant than in DNA replication.
The process of transcription done by RNA polymerases is controlled by the places that define the promoter (start point) and terminator (end point). Some other words that we usually use to define these regions are: proximal region (closer to promoter, upstream) and distal region, (closer to the terminator, downstream). These regions are transcriptional unit segments that are going to be transcribed into RNA.
RNA polymerases recognize the promoter region and start to copy one strand of DNA into RNA by putting complementary bases. It keeps adding nucleotides ate the 3’ end until it finds the terminator. The reaction is almost the same that the one in DNA. The enzyme has a cavity that allows/fits the template strand of DNA and will allow RNA to exit.
We can summarize transcription in 4 steps: 1) Template recognition: RNA polymerase has to recognize the promoter. The two strands of DNA are going to separate by a helicase activity 2) Initiation: the RNA polymerase adds the first nucleotides complementary to the template strand 3) Elongation: from this point on, the RNA polymerase moves in Molecular biology – lecture 5 5’ 3’ direction and elongates the RNA copying information from the template strand.
Topoisomerases will prevent the supercoiling of the DNA strands 4) Termination: the RNA polymerases reads the terminator sequence and stops adding nucleotides Most illnesses related to the transcription process occur during template recognition and initiation.
2.1. PROMOTERS IN PROKARYOTES The sequence of the promoter is special and it founds in -10(TATAAT) and -35(TTGACA), the numbers are relative to the number of nucleotides upstream from the initiation point.
These two sequences are found in most bacterial genes but there’s variability. Both sequences are called consensus sequences because is the more common. In the case of the -10 sequence, the first T is 80% always there and the following A is not present in only 5% of the promoters. From the energetic point of view, the presence of specific bases in the -10 sequence allows a stronger interaction with the RNA polymerase. If many bases are absent of it, the RNA polymerase will not be able to bind (and detect) the promoter sequence and the gene won’t be expressed. In a laboratory, if we want high levels of expression we will create a promoter with the same nucleotides of the -10 and -35 sequences and transcription will be more efficient, but if we want to reduce the expression level of a particular gene, you will change the bases so the RNA polymerase will have problems to detect the promoter region and the transcription will be less efficient. The close is the sequence to the consensus sequence, the more efficient the process is. In prokaryotes, RNA polymerase needs sigma subunits because these subunits are the ones that recognize the promoter.
Molecular biology – lecture 5 2.2. PROMOTERS IN EUKARIOTES In eukaryotes, RNA polymerase recognizes the promoter by many additional proteins called general transcription factors because they can be found in almost all eukaryotic genes. At the promoter we find an important sequence in 20 (TATA). This sequence is present in almost all of our genes but there are also other necessary sequences called enhancers (intensificadores). The proteins that cut these enhancers are called specific transcription factors because they are specific for different tissues. Ex: our liver cells are different because they express different enhancers. Enhancers are recognized by specific transcriptional factors that will only operate in those genes having the enhance sequence.
Because of the complexity of eukaryotes, we can find 3 different RNA polymerases depending on the genes that they describe: 1) RNA polymerase II: takes care of almost all of our genes, synthesizes the precursor of our genes and produces de basal complex.
2) RNA polymerases I and III: produce rRNAs, tRNAs and snRNAs The protein that recognizes the TATA sequence is the TFIID. This protein is a complex of polypeptides. Once TFIID is bound to the promoter, it will drive the assembly of the basal complex. The basal complex is not sufficient for transcription and needs the work of the specific transcription factors. The contact of these specific transcription factors with the basal complex releases the RNA polymerase to start RNA synthesis.
2.3. TERMINATOR IN PROKARYOTES At the end of the bacterial DNA, a region is able to fold back (make a loop) and make a strong structure. This loop will block the elongation process and prevent the RNA polymerase to go further ahead. The loop will block transcription and once the RNA polymerase is arrested by the loop, it all depends on the interaction with RNA and DNA. If this region contains many A-U pairs the DNA-RNA interaction is very unstable (because A Molecular biology – lecture 5 interacts with U with only two hydrogen bonds) and it finally dissociate. This is called an intrinsic terminator. Some other genes in bacteria need the help of a protein called Rho. Rho binds to RNA and moves throw it until it arrives to the RNA polymerase and helps the complex to dissociate. In these cases, the interaction between DNA and RNA is more stable because in this region of the RNA there are no Us. These terminators are called rho-dependent terminators.
2.4. TERMINATOR IN EUKARIOTES We don’t know a lot but it seems that most of the genes use the intrinsic termination 3. IMPORTANT SIFFERENCES BETWEEN DNA AND RNA As DNA, RNA is a linear polymer mode of four different types of nucleotide subunits linked together by phosphodiester bonds. The two differences on the structure are: 1) The nucleotides in RNA are ribonucleotides. This means that they contain the sugar ribose rather than deoxyribose. The chemical structure of the ribose contains an oxygen group at C2’ that may help breaking the phosphodiester bond, and thus makes RNA less stable than DNA.
2) RNA contains the base uracil instead of thymine. Uracil, as thymine, can base-pair by hydrogen bonds with adenine and also with guanine Molecular biology – lecture 5 Although these differences are slight, the structure of both DNA and RNA differ a lot in overall structure: 1) RNA doesn’t have a complementary strand and makes intramolecular paring with itself. RNA is single stranded and can therefore fold up into a particular shape, just as polypeptide chain. This ability to fold up into complex three-dimensional shapes allows some RNA molecules to have precise structural and catalytic functions.
2) As the oxygen groups on the ribose need to be stabilized, they interact with water.
There is some water in RNA 4. KINDS OF RNA mRNA (messenger): brings the protein code tRNA (transfer): important for translation rRNA(ribosomal): important for translation hnRNA (heterologous nuclear): precursor of mRNA snRNA (small nuclear): important for processing hnRNA into mRNA ...