DeVry University, Chicago BIOS 390 ERIC KYLE week 5 & 6 Labs BIOS390.

WEEK 5 & 6: LAB BIOS390 ERIC KYLE WEEK 5: LAB: transcription in prokaryotes Transcription is the first step in gene expression. It involves copying a gene's DNA sequence to make an RNA molecule. Before transcription can take place, the DNA double helix must unwind near the gene that is getting transcribed. The region of opened-up DNA is called a transcription bubble. Transcription uses one of the two exposed DNA strands as a template; this strand is called the template strand. The RNA product is complementary to the template strand and is almost identical to the other DNA strand, called the nontemplate (or coding) strand. However, there is one important difference: in the newly made RNA, all the T nucleotides are replaced with U nucleotides. The site on the DNA from which the first RNA nucleotide is transcribed is called the +1 site, or the initiation site. Nucleotides that come before the initiation site are given negative numbers and said to be upstream. Nucleotides that come after the initiation site are marked with positive numbers and said to be downstream. RNA polymerases are enzymes that transcribe DNA into RNA. Using a DNA template, RNA polymerase builds a new RNA molecule through base pairing. RNA polymerase always builds a new RNA strand in the 5’ to 3’ direction. That is, it can only add RNA nucleotides (A, U, C, or G) to the 3' end of the strand. RNA polymerases are large enzymes with multiple subunits, even in simple organisms like bacteria. Transcription initiation To begin transcribing a gene, RNA polymerase binds to the DNA of the gene at a region called the promoter. Basically, the promoter tells the polymerase where to "sit down" on the DNA and begin transcribing. In bacteria, each group of genes transcribed together has its own promoter. A promoter contains DNA sequences that let RNA polymerase, or its helper proteins attach to the DNA. Once the transcription bubble has formed, the polymerase can start transcribing. A typical bacterial promoter contains two important DNA sequences, the -10 and -35 elements. RNA polymerase recognizes and binds directly to these sequences. The sequences position the polymerase in the right spot to start transcribing a target gene, and they also make sure it's pointing in the right direction. Once the RNA polymerase has bound, it can open up the DNA and get to work. DNA opening occurs at the -10 element, where the strands are easy to separate due to the many As and Ts (which bind to each other using just two hydrogen bonds, rather than the three hydrogen bonds of Gs and Cs). The -10 and the -35 elements get their names because they come 35 and 10 nucleotides before the initiation site (+1 in the DNA). The minus signs just mean that they are before, not after, the initiation site. Elongation Once RNA polymerase is in position at the promoter, the next step of transcription—elongation —can begin. Basically, elongation is the stage when the RNA strand gets longer, thanks to the addition of new nucleotides. During elongation, RNA polymerase "walks" along one strand of DNA, known as the template strand, in the 3' to 5' direction. For each nucleotide in the template, RNA polymerase adds a matching (complementary) RNA nucleotide to the 3' end of the RNA strand. The RNA transcript is nearly identical to the non-template, or coding, strand of DNA.