Bacterial Genetics: Lecture # 05: Transcription

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BACTERIAL GENETICS

Lecture # 05: Transcription


Introduction

• Transcription is the synthesis of an RNA molecule from a DNA


template.

• All cellular RNAs are synthesized from DNA templates through the
process of transcription.

• Transcription is in many ways similar to the process of replication, but


a fundamental difference relates to the length of the template used.
Introduction (contd.)

• In replication, all the nucleotides in the DNA molecule are copied, but
in transcription, only parts of the DNA molecule are transcribed into
RNA.

• Because not all gene products are needed at the same time or in the
same cell, the constant transcription of all of a cell’s genes would be
highly inefficient.
Introduction (contd.)

• Furthermore, much of the DNA does not encode any functional


product, and transcription of such sequences would be pointless.

• Transcription is, in fact, a highly selective process: individual genes are


transcribed only as their products are needed.

• Like replication, transcription requires three major components:


Introduction (contd.)

• 1. A DNA template

• 2. The raw materials (ribonucleotide triphosphates) needed to build a


new RNA molecule

• 3. The transcription apparatus, consisting of the proteins necessary


for catalyzing the synthesis of RNA
The Template
• In 1970, Oscar Miller, Jr., Barbara Hamkalo, and Charles Thomas used
electron microscopy to demonstrate that RNA is transcribed from a
DNA template.

• They broke open cells, extracted chromatin, and spread the


chromatin onto a fine mesh grid.

• Under the electron microscope, they observed Christmas-tree-like


structures, each consisting of a thin central fiber (the trunk of the
tree) to which were attached strings (the branches) bearing granules.
The Template (contd.)
• The addition of deoxyribonuclease (an enzyme that degrades DNA)
caused the central fibers to disappear, indicating that the “tree
trunks” were DNA molecules.

• Ribonuclease (an enzyme that degrades RNA) removed the granular


strings, indicating that the branches were RNA.

• Their conclusion was that each “Christmas tree” represents a gene


undergoing transcription
The Template (contd.)

• The transcription of each gene begins at the top of the tree; there,
little of the DNA has been transcribed, and the RNA branches are
short.

• As the transcription apparatus moves down the tree, transcribing


more of the template, the RNA molecules lengthen, producing the
long branches at the bottom.
The Template (contd.)
• THE TRANSCRIBED STRAND:

• The template for RNA synthesis, as for DNA synthesis, is a single strand of
the DNA double helix.

• Unlike replication, however, the transcription of a gene takes place on only


one of the two nucleotide strands of DNA.

• The nucleotide strand used for transcription is termed the template strand.
The Template (contd.)

• The other strand, called the nontemplate strand, is not ordinarily


transcribed.

• Thus, within a gene, only one of the nucleotide strands is normally


transcribed into RNA.

• During transcription, an RNA molecule that is complementary and


antiparallel to the DNA template strand is synthesized.
The Template (contd.)
• The RNA transcript has the same polarity and base sequence as the
nontemplate strand, except that it contains U rather than T.

• In most organisms, each gene is transcribed from a single DNA strand, but
different genes may be transcribed from different strands.

• TRANSCRIPTION UNIT:

• A transcription unit is a stretch of DNA that encodes an RNA molecule and


the sequences necessary for its transcription.
The Template (contd.)

• Included within a transcription unit are three critical regions: a


promoter, an RNA-coding region, and a terminator.

• The promoter is a DNA sequence that the transcription apparatus


recognizes and binds.

• The promoter indicates which of the two DNA strands is to be read as


the template and the direction of transcription.
The Template (contd.)
• It also determines the transcription start site, the first nucleotide that
will be transcribed into RNA.

• In many transcription units, the promoter is located next to the


transcription start site but is not itself transcribed.

• The second critical region of the transcription unit is the RNA-coding


region, a sequence of DNA nucleotides that is copied into an RNA
molecule.
The Template (contd.)
• The third component of the transcription unit is the terminator, a
sequence of nucleotides that signals where transcription is to end.

• Terminators are usually part of the RNA-coding sequence; transcription


stops only after the terminator has been copied into RNA.

• Molecular biologists often use the terms upstream and downstream to


refer to the direction of transcription and the locations of nucleotide
sequences surrounding the RNA-coding region.
The Template (contd.)

• The transcription apparatus is said to move downstream during


transcription: it binds to the promoter (which is usually upstream of
the transcription start site) and moves toward the terminator (which
is downstream of the start site).

• When DNA sequences are written out, often the sequence of only
one of the two strands is listed.
The Template (contd.)
• Molecular biologists typically write the sequence of the non-template
strand because it will be the same as the sequence of the RNA
transcribed from the template strand (with the exception that U in
RNA replaces T in DNA).

• By convention, the sequence of the non-template strand is written


with the 5′ end on the left and the 3′ end on the right.

• The first nucleotide transcribed (the transcription start site) is


numbered +1.
The Template (contd.)
• Nucleotides downstream of the start site are assigned positive
numbers, and nucleotides upstream of the start site are assigned
negative numbers.

• So, nucleotide +34 would be 34 nucleotides downstream of the start


site, whereas nucleotide -75 would be 75 nucleotides upstream of the
start site.

• There is no nucleotide numbered 0.


The Substrate for Transcription

• RNA is synthesized from ribonucleoside triphosphates (rNTPs), each


consisting of a ribose sugar and a base (a nucleoside) attached to
three phosphate groups.

• In RNA synthesis, nucleotides are added one at a time to the 3′-OH


group of the growing RNA molecule.
The Substrate for Transcription (contd.)

• Two phosphate groups are cleaved from the incoming ribonucleoside


triphosphate; the remaining phosphate group participates in a
phosphodiester bond that connects the nucleotide to the growing
RNA molecule.

• The overall chemical reaction for the addition of each nucleotide is


The Substrate for Transcription (contd.)
• where PPi represents pyrophosphate.

• Nucleotides are always added to the 3′ end of the RNA molecule, and
the direction of transcription is therefore 5′→3′.

• Thus, the newly synthesized RNA is complementary and antiparallel


to the template strand.

• Unlike DNA synthesis, RNA synthesis does not require a primer.


The Transcription Apparatus

• The action of RNA polymerase is enhanced by a number of accessory


proteins that join and leave the polymerase at different stages of the
process.

• Each accessory protein is responsible for providing or regulating a


special function.
The Transcription Apparatus (contd.)

• BACTERIAL RNA POLYMERASE:

• Bacterial cells typically possess only one type of RNA polymerase,


which catalyzes the synthesis of all classes of bacterial RNA: mRNA,
tRNA, and rRNA.

• Bacterial RNA polymerase is a large multimeric enzyme (meaning that


it consists of several polypeptide chains).
The Transcription Apparatus (contd.)
• At the heart of most bacterial RNA polymerases are five subunits that
make up the core enzyme: two copies of a subunit called alpha (α)
and single copies of subunits beta (β), beta prime (β′), and omega (ω).

• The ω subunit is not essential for transcription, but it helps stabilize


the enzyme.

• The core enzyme catalyzes the elongation of the RNA molecule by the
addition of RNA nucleotides.
The Transcription Apparatus (contd.)

• Other functional subunits join and leave the core enzyme at particular
stages of the transcription process.

• The sigma (σ) factor controls the binding of RNA polymerase to the
promoter.

• Without sigma, RNA polymerase initiates transcription at a random


point along the DNA.
The Transcription Apparatus (contd.)

• After sigma has associated with the core enzyme (forming a


holoenzyme), RNA polymerase binds stably only to the promoter and
initiates transcription at the proper start site.

• Sigma is required only for promoter binding and initiation; after a few
RNA nucleotides have been joined together, sigma usually detaches
from the core enzyme.
The Transcription Apparatus (contd.)

• Many bacteria have multiple types of sigma factors; each type


initiates the binding of RNA polymerase to a particular set of
promoters.

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