Genetic Information Flow: RNA Structure Dr. Umut Fahrioglu, PhD MSc Introduction So far we have looked at DNA structure, chemistry, replication and its packaging into chromosomes. We will start to look at how the DNA is expressed, which means we will explore how the information coded by the DNA is used by the cell to specify RNA and protein molecules. DNA is like a book. We already know the alphabet (A, C, T and G). The specific linear order of these four bases is the genetic information. The information can be looked at in subsets called genes, which are Mendels units of inheritance One gene: one enzyme hypothesis -Neurospora experiment by Tatum and Beadle. -Isolated mutants with genetic blocks essential for growth. -80,000 spores tested. -A mutation can be found for every enzymatic reaction. One gene encodes one polypeptide: Human haemoglobin study Central Dogma of Molecular Biology By Francis Crick The process is compartmentalized Sense vs anti-sense strand Genetic Information Flow Exceptions to the Central Dogma DNA retroviruses use reverse transcriptase to replicate their genome (David Baltimore and Howard Temin) RNA viruses mRNA introns (splicing) (Philip Sharp and Richard Roberts) RNA editing (deamination of cytosine to yield uracil in mRNA) RNA interference (RNAi) a mechanism of post-transcriptional gene silencing utilizing double-stranded RNA RNAs (ribozymes) can catalyze an enzymatic reaction (Thomas Cech and Sidney Altman) RNA Protein Prions are heritable proteins responsible for neurological infectious diseases (e.g. scrapie and mad cow) (Stanley Pruisner) Epigenetic marks, such as patterns of DNA methylation, can be inherited and provide information other than the DNA sequence Types of RNA mRNA is the type of RNA that will be translated into a protein. It carries the genetic message from the DNA to the ribosome. It specifies the order of amino acids that need to be put together in a linear order. Amino acid sequence in turn dictates how each protein folds and to give a specific molecule with a distinctive shape and chemistry. rRNA is an integral part of a the ribosome. tRNA becomes important for translating the message into amino acid sequence. They deliver amino acids to the ribosome. Genes can be expressed with different efficiencies One gene can be transcribed and translated much more efficiently than another. This allows for the amount of that gene to be greater than the other. Many identical RNA copies can be made from the same gene and each RNA can direct the synthesis of many identical protein molecules. This allows the cell to synthesize large amounts of the protein rapidly if needed. A cell can regulate the expression of each of its genes according to the needs of the moment. It does this by controlling its RNA production most of the time. RNA vs. DNA RNA terminology nucleoside base sugar BaseNucleoside (RNA)Deoxynucleoside (DNA) AdenineAdenosineDeoxyadenosine GuanineGuanosineDeoxyguanosine CytosineCytidineDeoxycytidine UracilUridine(not usually found) Thymine(not usually found)(Deoxy)thymidine RNA structure 1. RNA can be single or double stranded 2. G-C pairs have 3 hydrogen bonds 3. A-U pairs have 2 hydrogen bonds 4. Single-stranded, double-stranded, and loop RNA present different surfaces mRNA Structure mRNAs are single stranded RNA molecules. Sometimes 2000 nucleotides long. They are copied from the TEMPLATE strand of the DNA, to produce the SENSE strand in RNA They are transcribed from the 5 to the 3 end They are translated from the 5 to the 3 end Generally mRNAs are linear (although some prokaryotic RNA viruses are circular and act as mRNAs) 5 cap and 3 poly(A) tail of mRNA Prokaryotic vs. Eukaryotic mRNA In prokaryotes, mRNAs are polycistronic which means they can code for more than one protein product (But they can also just code for one). In eukaryotes, mRNAs are monocistronic which means that they only encode one protein. There are some eukaryotic viruses which have polyproteins but these proteins are produced as a single peptide chain and are cleaved into separate proteins after translation. mRNA lifecycle mRNA is synthesised by RNA Polymerase Translated (once or many times) Degraded by RNAses Steady state level depends on the rates of both synthesis and degradation Comparison of mRNA Prokaryotic mRNAEukaryotic mRNA Linear RNA structure 5 and 3 ends are unmodified Ribosomes bind at ribosome binding site, internally within mRNA (do not require a free 5 end) Can contain many open reading frames (ORFs) Translated from 5 end to 3 end Transcribed and translated together Linear RNA structure 5-cap and 3-poly(A) tail Poly(A) tail is used by the ribosome for recognition. Usually contains only one ORF. Translated from 5 to 3 end Transcribed, processed, transported to the cytoplasm and then translated. Transfer RNA (tRNA) They are small RNAs in which the mature tRNA are 70 (75) - 90 nucleotides in length. The precursor can be around 126 nucleotides. Some of the nucleotides are chemically modified. Some examples are hypoxanthine, ribotymine and pseudouridine. They are created after transcription. Because of the complementary sequences within the linear RNA they can form highly conserved secondary (hairpins) and tertiary structures (where the structure is folded to form an L). Each class of tRNA is charged with a single amino acid. tRNA tyr indicates the tRNA carrying the amino acid tyrosine (cognate amino acid). Each tRNA has a specific trinucleotide anti-codon for mRNA recognition. There is a conservation of structure and function in both prokaryotes and eukaryotes. General structural features of tRNA Cloverleaf secondary structure with intramolecular base-pairing A loop that contains the anti- codon (in the 3 to 5 direction), called the anti-codon loop. At the 3 end all tRNAs contain CCA. This is the end where the amino acid is covalently linked to the adenosine residue. All tRNA contain the nucleotide G at the 5 end. The lengths of the loops and stems are very similar among all the tRNA. Charging the tRNA Ribosomal RNA (rRNA) By far the most stable and the most abundant form of RNA in the cells Represents about 70-80% of total RNA (tRNA is about 10-20% and mRNA less than 10%). Eukaryotic cells have 4 species of rRNA based on sedimentation upon centrifuge. They are referred to with their sedimentation coefficients (S). The three larger rRNA species are produced as a single transcript. Multiple copies of the three large rRNA transcription units are present in eukaryotic cells ( copies divided among 5 chromosomes). Transcribed by RNA polymerase I. The small rRNA is separate and is transcribed by RNA polymerase III. NOR part of the nucleolus.