WEEK 5 & 6: LAB BIOS390
BIOS 390 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
BIOS 390 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
