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BIOLOGY 1A03 Lecture Notes - Aminoacyl Trna Synthetase, Rna Polymerase Iii, Aminoacyl-Trna


Department
Biology
Course Code
BIOLOGY 1A03
Professor
Lovaye Kajiura

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Biology Chapter 16: Transcription and Translation
Transcription in Bacteria
The first step in converting genetic information into proteins is the synthesis of a
messenger RNA version of the instructions archived in DNA
Enzymes called RNA polymerases are responsible for synthesizing mRNA
Like DNA polymerases, an RNA polymerase performs a template-directed synthesis in
the 5’ 3’ direction
Unlike DNA polymerases, RNA polymerases do not require a primer to begin
transcription
Transcription occurs when RNA polymerase matches the base in a ribonucleotides
triphosphate with the complementary base in a gene (a section of DNA that codes for a
protein or RNA)
Once a matching ribonucleotides is in place, RNA polymerase catalyzes the formation of
a phosphodiester bond between the 3’ end of the growing mRNA chain and the new
ribonucleotides
As this matching-and-catalysis process continues, an RNA that is complementary to the
gene is synthesized
Only one of the two DNA strands is used as a template and transcribed, called the
template strand
The other strand is called the non-template strand, also known as the coding strand
because its sequence matches the sequence of RNA that is transcribed from the
template strand and codes for a polypeptide
Key difference is that RNA has uracil (U) rather than thymine (T)
RNA Polymerase Structure and Function
o To understand structure, biologists used X-ray crystallography, which allowed
them to obtain information about the 3D structure
o Results indicate that the enzyme is large and globular and has several prominent
channels running through the interior
o The enzyme’s active site, where the phosphodiester bonds form, is located
where several of these channels intersect
Initiation: How Does Transcription Begin
o Researchers discovered that the enzyme cannot initiate transcription on its own
o Instead, a detachable protein subunit called sigma must bind to RNA polymerase
before transcription can begin
Sigma is a protein that recognizes promoter regions
o RNA polymerase and sigma form a holoenzyme, which consists of a core enzyme,
which contains the active site for catalysis, and other required proteins
o When researchers mixed RNA polymerase, sigma and DNA together, they found
that the holoenzymes bound tightly to specific sections of DNA

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o These binding sites were named promoters because they are sections of DNA
where transcription begins
o The discovery of promoters suggests that sigma’s function is regulatory in nature
o Sigma appeared to be responsible for guiding the RNA polymerase to specific
locations where transcription should begin
o Results showed that promoters were located on the non-template strand, were
40-50 bp long and had a particular section that looked similar
o This similar segment of DNA had a series of bases identical or similar to TATAAT
o This six base pair sequence is now known as the -10 box because it is centred
about 10 bases from the point where RNA polymerase starts transcription
o DNA that is located in the direction RNA polymerase moves during transcription
is said to be downstream
o DNA in the opposite direction is said to be upstream, thus the -10 box is centred
10 bases upstream from the transcription start site
o The place where transcription begins is the +1 site
o Researchers also recognized that the sequence TTGACA occurred in these same
promoters, centred about 35 bases upstream from the +1 site
o This second key sequence is called the -35 box
o Follow up work showed that transcription begins when sigma binds to the -35
and -10 boxes
o Supports the hypothesis that sigma is a regulatory protein
o Sigma tells RNA polymerase where and when to start synthesizing RNA
o Once sigma binds to a promoter, the DNA helix opens and creates two strands of
single-stranded DNA
o The template strand is threaded through a channel that leads to the active site
inside RNA polymerase
o Monomers known as ribonucleoside triphosphate (NTPs) enter a channel at the
bottom of the enzyme and diffuse to the active site
o When an incoming NTP pairs with a complementary base on the template strand
of DNA, RNA polymerization begins
o Sigma is released once RNA synthesis is under way
Elongation
o During the elongation phase of transcription, RNA polymerase moves along the
DNA template strand in the 3’ 5’ direction of the template strand,
synthesizing RNA in the 5’ 3’ direction
o In the interior of the enzyme, a group of projecting AA called the enzyme’s
zipper helps open the double helix at the upstream end and a nearby group of
AA called the rudder steers the template and non-template strands through
channels inside the enzyme
o Meanwhile, the enzyme’s active site catalyzes the addition of nucleotides to the
3’ end of the growing RNA molecule at the rate of about 50 nucleotides/second
o During elongation, all the prominent channels or grooves in the enzyme are filled
o Double stranded DNA goes into and out of one groove, NTPs enter another and
the growing RNA strand exits to the rear

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Termination
o In most cases, transcription stops when RNA polymerase reaches a stretch of
DNA sequence that functions as a transcription termination signal
o The bases that make up a termination signal code for a stretch of RNA with an
unusual property, as soon as it is synthesized, the RNA sequence folds back on
itself and forms a short double helix that is held together by complementary
base pairing
o The secondary structure that results is called a hairpin
o The formation of the hairpin structure is thought to disrupt the interaction
between RNA polymerase and the RNA transcript, resulting in the physical
separation of the enzyme and its product
Summary
o Transcription begins when sigma, as part of the holoenzyme complex, binds to
the promoter at the start of a gene
o Once binding occurs, RNA polymerase begins to synthesize mRNA by adding
ribonucleotides that are complementary to the template strand in DNA
o Transcription ends when a termination signal at the end of the gene leads to the
formation of hairpin in mRNA, disrupting the transcription complex
Transcription and RNA Processing in Eukaryotes
Transcription in eukaryotes similar to bacterial transcription in that RNA polymerase
does not bind directly to promoter sequences by itself
Instead, proteins called basal transcription factors initiate eukaryotic transcription by
matching the enzyme with the appropriate promoter region in DNA
The function of basal transcription factors is analogous to the function of the sigma
proteins in bacteria, except that BTF interact with DNA independent of RNA polymerase
Research showed several important distinctions about how transcription works in
bacteria and eukaryotes
o In bacteria a single sigma protein binds to a promoter and initiates transcription,
but in eukaryotes many BSL are required to initiate transcription
In eukaryotes the machinery required to start transcription is complex
o Eukaryotes have three distinct types of RNA polymerase, instead of just one
RNA pol I transcribes genes that code for most of the large RNA
molecules found in ribosomes
RNA pol II transcribes protein-coding genes (produces mRNAs)
RNA pol III transcribes genes that code for tRNA and genes that code for
one of the small RNA molecules found in ribosomes
RNA pol II and pol III transcribe RNA molecules found in snRNPs
o Although eukaryotic genomes contain promoters that signal where transcription
should begin, just as bacteria do, the promoters in eukaryotic DNA are much
more diverse and complex than bacterial promoters
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