Transcription and the Central Dogma 1961: DNA is the molecule which stores genetic information, but DNA is in nucleus, ribosomes (where protein synthesis takes place) are in the cytoplasm. RNA is synthesized in the nucleus, is similar to DNA. RNA migrates to cytoplasm (where ribosomes are) Amount of RNA generally proportional to amount of proteins in the cell. All this suggests role of RNA as messenger.

About RNA 1) DNA is double stranded, but RNA is single stranded. However, RNA can base-pair with itself to create double stranded regions. RNA DNA tRNA genetics.gsk.com/graphics/ dna-big.gif http://www.fhi-berlin.mpg.de/th/JG/RNA.jpg http://www.santafe.edu/images/rna.gif

About RNA-2 2) RNA contains ribose instead of deoxyribose 3) RNA contains uracil instead of thymine. www.layevangelism.com/.../ deoxyribose.htm http://www.rothamsted.bbsrc.ac.uk/notebook/courses/guide/images/uracil.gif

3 kinds of RNA mRNA: a copy of the gene; is translated to make protein. tRNA: smallest RNA, does actual decoding. rRNA: 3 sizes that, along with proteins, make up a ribosome. tRNA rRNA http://www.cu.lu/labext/rcms/cppe/traducti/tjpeg/trna.jpeg; Tobin and Duschek, Asking About Life; http://www.tokyo-ed.ac.jp/genet/mutation/nort.gif

More kinds of RNA snRNAs snoRNAs Other RNAs, e.g. Xist Small RNAs (100-200 bases) that attach to proteins to form snRNPs (small nuclear ribonucleoproteins) snRNPs are components of the spliceosome Removes introns from pre-mRNA snoRNAs Small nucleolar RNAs, process rRNA Modify other RNAs; guide RNA of telomerase Other RNAs, e.g. Xist

All RNA comes from the DNA mRNA, obviously tRNA, rRNA Telomerase guide RNA Xist antisense-RNA Thus, we talk about RNA genes, sections of DNA that code for RNAs

Transcription: making mRNA RNA a polymer assembled from monomers Ribonucleoside triphosphates: ATP, UTP, GTP,CTP RNA polymerase Multi-component enzyme Needs a template, but NOT a primer In bacteria, a component (sigma) recognizes the promoter as the place on DNA to start synthesis Synthesis proceeds 5’ to 3’, just as in DNA mRNA is complementary and antiparallel to the DNA strand being copied.

Transcription-2 The order of nucleotides in the RNA reflects the order in the DNA If RNA is complementary to one DNA strand, then it is identical (except for T change to U) to the other DNA strand. Either DNA strand may contain the gene! Transcription just runs the other direction.

Sense, antisense Compare the sense strand of the DNA to the mRNA. Note that mRNA synthesis will be 5’ to 3’ and antiparallel. http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/S/SenseStrand.gif

More about RNA polymerase In bacteria, components are ββ’α2φσ RNA polymerase is processive; once enzyme attaches to DNA, it can copy >10,000 nucleotides without falling off. In eukaryotes, there are 3 RNA polymerases: One for rRNA One for tRNAs and some rRNA One for all mRNAs and some small RNAs (involved in RNA processing)

Transcription needs a Promoter A promoter is non-transcribed DNA Prokaryotes Eukaryotes http://opbs.okstate.edu/~petracek/2002%20Gene%20expression/img043.gif

The Process of Transcription Promoter recognition: 2 consensus sequences The -10 region: TATAAT (10 bases upstream from where transcription actually starts. The -35 region, farther upstream, also important. “Consensus” sequence meaning the DNA sequence from many genes averages out to this. The closer these 2 regions actually are to the consensus sequences, the “stronger” the promoter, meaning the more likely RNA polymerase binding and transcription will occur.

Consensus sequence Numbers indicate the percentage of different genes in which that nucleotide appears in that spot in the promoter sequence. http://www.uark.edu/campus-resources/mivey/m4233/promoter.gif

The Process of Transcription-2 After binding to the promoter, polymerase “melts” DNA, lines up first base at the +1 site = Initiation. RNA synthesis continues (Elongation), only the template strand being transcribed. Termination: must be a stop sign, right? In bacteria, hairpin loop followed by run of U’s in the RNA. Of course, the DNA must code for complementary bases and a run of A’s. See next. Termination factor “rho”. Accessory protein.

Termination of Transcription in Bacteria The hairpin loop destabilizes the interactions between the DNA, mRNA, and polymerase; U-A basepairs are very weak, and the complex falls apart. In euks, termination occurs with a processing step. http://www.blc.arizona.edu/marty/411/Modules/Weaver/Chap6/Fig.0649ac.gif

About mRNA structure, etc. Start site of transcription is NOT equal to start site of Translation First codon read, AUG, is downstream from the first ribonucleotides. +1 is transcription start, not translation start. AUG marks the beginning of an Open Reading Frame (ORF). Lifetime of a eukaryotic mRNA is variable For prokaryotes, mRNA is short lived, fits in with need of microbes to respond quickly to changes in environment.

Eukaryotic transcription Occurs in nucleus, then mRNA goes to cytoplasm Promoters but also enhancers Enhancers also segments of DNA Eukaryotic RNA requires processing Pre-mRNAs found in nucleus as hnRNA Heterogeneous nuclear RNA hnRNPs: heterogeneous nuclear ribonucleoproteins

Initiation of transcription (euks) Goldberg-Hogness (TATA) box at -35 CAAT box at -80 (non specific, but important) GGCCAATCT consensus sequence Enhancers Elements of DNA that promote transcription Can be upstream, downstream, even in gene

Eukaryotic RNA polymerase A 10 subunit machine Makes several attempts, abortive transcription Key: successful synthesis of short DNA-RNA hybrid, stabilizes association of Pol with DNA Then, processive to a high degree, transcribes until knocked off by termination signal.

Processing of mRNA Cap 7-methyl guanosine is added as a cap, 5’ to 5’ Cap aids in binding of mRNA to the ribosome Shine-Delgarno seq. does same in prokaryotes http://www.blc.arizona.edu/Marty/429/Lectures/Figures/CAP.GIF

Processing of mRNA-2 Poly-A tail In several steps, end of mRNA is cleaved off, and several rounds of AAAAAAA are added Poly-A tail improves stability of mRNA, resists degradation by nucleases in cell. departments.oxy.edu/.../ processing_of_hnrnas.htm

Introns and Exons Introns are intervening sequences that do not contain information for making the protein. Exons are the coding sequences left behind. http://www.emc.maricopa.edu/faculty/farabee/BIOBK/exintrons.gif

Removal of Introns Three types of intron-removal mechanisms Spliceosomes: hnRNA or pre-mRNA (to be processed) snRNPs: small nuclear ribonucleoproteins snRNAs: involved structurally and catalytically In some systems, intron codes for protein that splices out the intron! In Tetrahymena, intron is self-splicing: ribozyme

Splicing of Introns In most systems, snRNPs cut mRNA, introns come out, and snRNPs help splice exons back together. Particular sequences in the mRNA mark the beginning, end of exons and introns so snRNPs can do their job. http://www.plantsci.cam.ac.uk/Haseloff/SITEGRAPHICS/splice1.GIF

Introns: How were they discovered? RNA-DNA hybrids weren’t colinear- loops of DNA extend out where there is no RNA to base pair with it. RNA = red. Mutations in introns: don’t have much affect unless: Mutation is near a splice site Mutation is in a regulatory region (which could be in an intron) There is a separate gene within the intron.

Why are there introns? Very ancient? Located in the same positions in genes common to plants and animals. Maybe bacteria had them once and lost them. Self-splicing RNAs may be related to RNA as the first nucleic acid, a popular idea in evolution. Exon-shuffling: a model for gene evolution Some proteins fold into connected, functional sections called domains; these correspond to exons Perhaps exons were copied, shuffled to create new genes. Several human genes share exons.

Relationship between protein domains and exons http://www.mrc-lmb.cam.ac.uk/genomes/cvogel/SupraDomains/Data_new/sd.figure.supra-domain.jpg