Prokaryotic Transcription and RNA Modification Vocabulary

Chapter 12: Transcription and RNA Modification

Gene Transcription and RNA Modification

Introduction

• Transcription is the process of copying a DNA sequence into an RNA sequence.

• The DNA structure remains unaltered and continues to store information.

Gene Expression

• Protein-coding genes (structural genes) code for the amino acid sequence of a polypeptide.

• Transcription of a protein-coding gene produces messenger RNA (mRNA).

• The mRNA base sequence determines the amino acid sequence of a polypeptide during translation.

• One or more polypeptides constitute a protein.

• The synthesis of functional proteins determines an organism’s traits.

The Central Dogma of Genetics Terminology

• DNA replication: Makes DNA copies transmitted from cell to cell and from parent to offspring.

• Chromosomal DNA: Stores information in units called genes.

• Transcription: Produces an RNA copy of a gene.

• Messenger RNA (mRNA): A temporary copy of a gene that contains information to make a polypeptide.

• Translation: Produces a polypeptide using the information in mRNA.

• Polypeptide: Becomes part of a functional protein that contributes to an organism's traits.

Central Dogma

• DNA replication makes DNA copies transmitted from cell to cell and from parent to offspring.

• Chromosomal DNA stores information in units called genes.

• Transcription produces an RNA copy of a gene.

• Messenger RNA is a temporary copy of a gene that contains information to make a polypeptide.

• Translation produces a polypeptide using the information in mRNA.

• A polypeptide becomes part of a functional protein that contributes to an organism's traits.

12.1 Overview of Transcription

• DNA base sequences define the beginning and end of a gene and regulate the level of RNA synthesis.

• Proteins must recognize and act on DNA for transcription to occur.

• Gene expression is the overall process by which the information within a gene is used to produce a functional product which, in concert with environmental factors, can determine a trait.

Bacterial Gene and its mRNA

• Key components:

  • Regulatory sequence

  • Promoter

  • Terminator

  • Start codon

  • Stop codon

  • Ribosome-binding site

Organization of Sequences of a Bacterial Gene and Its mRNA Transcript

• Regulatory elements: Site for the binding of regulatory transcription factor proteins; these proteins influence the rate of transcription. They can be found in a variety of locations.

• Promoter: Site for RNA polymerase binding; signals the beginning of transcription.

• Terminator: Signals the end of transcription.

• Ribosome-binding site: Site for ribosome binding to mRNA in bacteria; translation begins near this site in the mRNA. In eukaryotes, the ribosome binds to a 7-methylguanosine cap in the mRNA, and the ribosome scans the RNA for a start codon.

Organization of Sequences of a Bacterial Gene and Its mRNA Transcript 3

• Codons: 3 nucleotide sequences within the mRNA that specify particular amino acids. The sequence of codons within mRNA determines the sequence of amino acids within a polypeptide.

• Start codon: Specifies the first amino acid in a polypeptide sequence, usually a formyl methionine (in bacteria) or a methionine (in eukaryotes).

• Stop codon: Specifies the end of polypeptide synthesis.

• A bacterial mRNA may be polycistronic, which means it codes two or more polypeptides.

Gene Expression Requires Base Sequences

• The DNA strand that is actually transcribed (used as the template) is termed the template strand.

• The RNA transcript is complementary to the template strand.

• The opposite strand is called the coding strand or the sense strand, as well as the nontemplate strand.

• The base sequence in RNA is identical to the coding strand of DNA, except for the substitution of uracil (U) in RNA for thymine (T) in DNA.

Gene Expression Requires Base Sequences 2

• Transcription factors recognize the promoter and regulatory elements to control transcription.

• mRNA sequences such as the ribosomal-binding site and codons direct translation.

Template strand, Non-template strand and coding strand

• The opposite strand is called the non-template or coding strand or sense strand.

• The base sequence is identical to transcript.

Transcription

Transcription occurs in three stages:

• Initiation

• Elongation

• Termination

These steps involve protein-DNA interactions.

• Proteins such as RNA polymerase interact with DNA sequences.

RNA transcripts and their different functions

• Structural genes: Transcribed into mRNA; constitutes about 90% of all genes.

• Non-Structural genes: Not translated. Examples: Ribosomes, Spliceosomes, Signal Recognition Particle, Telomerase

Promoters 1

• Promoters are DNA sequences that “promote” gene expression.

• More precisely, they direct the exact location for the initiation of transcription.

• Promoters are typically located just upstream of the site where transcription of a gene actually begins.

• The bases in a promoter sequence are numbered in relation to the transcriptional start site.

Numbering system of promoters

• Includes the -35 sequence, -10 sequence, and the transcriptional start site (+1).

Promoters 2

• Promoters vary at the -35 and -10 sequences.

• The most common sequence is called the consensus sequence.

• The consensus sequence is likely to result in a high level of transcription.

• Sequences that deviate from the consensus sequence typically result in lower levels of transcription.

Examples of promoter sequences in bacteria

Bacterial RNA Polymerase

RNA polymerase is the enzyme that catalyzes the synthesis of RNA. In E. coli, the RNA polymerase holoenzyme is composed of:

• Core enzyme: Four subunits = \alpha_2\beta\beta’\omega

• Sigma factor: One subunit = \sigma

• These subunits play distinct functional roles.

Initiation of Transcription in Bacteria

• The RNA polymerase holoenzyme binds loosely to the DNA.

• It then scans along the DNA until it encounters a promoter region.

• When it does, the sigma factor recognizes both the –35 and –10 regions.

• A region within the sigma factor that contains a helix-turn-helix structure is involved in tighter binding to the DNA.

Initiation of Transcription (cont’d)

• The binding of the RNA polymerase to the promoter forms the closed complex.

• Then, the open complex is formed when the TATAAT box in the -10 region is unwound.

  • A-T bonds are more easily separated.

• A short RNA strand is made within the open complex.

• The sigma factor is released at this point.

  • This marks the end of initiation.

• The core enzyme now slides down the DNA to synthesize an RNA strand.

  • This is known as the elongation phase; no primer is required.

Initiation of Transcription

• RNA polymerase holoenzyme slides along the DNA, \sigma factor recognizes a promoter, and RNA polymerase holoenzyme forms a closed complex.

• An open complex is formed, and a short RNA is made.

• \sigma factor is released, and the core enzyme can proceed down the DNA, creating the RNA transcript.

Elongation of Transcript

• The open complex formed by the action of RNA polymerase is about 17 bases long.

• Behind the open complex, the DNA rewinds back into a double helix.

• On average, the rate of RNA synthesis is about 43 nucleotides per second!

• The figure here depicts the synthesis of an RNA transcript.

Synthesis of the RNA transcript

• RNA polymerase slides along the DNA, creating an open complex as it moves.

• RNA polymerase slides along the template strand in a 3’ to 5’ direction, and RNA is synthesized in a 5’ to 3’ direction using nucleoside triphosphates as precursors. Pyrophosphate is released.

• The complementary rule is the same as the AT/GC rule except that U is substituted for T in the RNA.

Termination of Bacterial transcription

• Termination is the end of RNA synthesis.

• It occurs when the short RNA-DNA hybrid of the open complex is forced to separate.

• This releases the newly made RNA as well as the RNA polymerase.

• E. coli has two different mechanisms for termination:

  1. rho-dependent termination

     • Requires a protein known as \\rho (rho)

  2. rho-independent termination

     • Does not require \\rho

Rho dependent Termination

• Rho utilizes a rut site.

\\rho-independent termination

• \\rho-independent termination is facilitated by two sequences in the RNA:

  1. A uracil-rich sequence located at the 3’ end of the RNA

  2. A stem-loop structure upstream of the uracil-rich sequence