Transcription in Prokaryotes: A Comprehensive Guide, Lecture notes of Molecular biology

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B.Sc. Biotechnology Molecular Biology

Transcription in Prokaryotes

Praveen Deepak

Assistant Professor of Zoology

Swami Sahjanand College

Jehanabad

Introduction

 It is the process of synthesis of messenger RNA transcripts by copying the template

strand of DNA in the prokaryotes. Messenger RNA is then translated to produce

proteins (some are not translated e.g. ncRNA or non-coding RNA).

 It occurs in the protoplasm/cytoplasm alongside translation (In eukaryotes,

translation can not occur simultaneously with the transcription).

 During replication entire genome is copied but in transcription only the selected

portion of genome , i.e. called as gene, is copied.

 The enzyme involved in transcription is RNA polymerase. Unlike DNA polymerase it

can initiate transcription by itself, it does not require primase (More exactly it is a

DNA dependent RNA polymerase).

 Like DNA replication, it is also a multistep process involving several enzymes and a

number of regulatory molecules, called transcription factor (some factors are

homodimers containing helix-turn-helix DNA-binding motifs).

 There are three steps in transcription:

o Initiation – Formation of Transcription Initiation Complex (TIC)

o Elongation – Formation of mRNA complementary to the DNA template

o Termination – Termination of transcription

Structure of gene  A " gene " occurs over a particular physical region called locus of dsDNA molecule.  It includes an RNA-coding region, where transcription occurs from the DNA template strand.  It runs between the initiation and termination sites in 5 ‘ to 3 ' direction.  Control regions for transcription, i.e., promoters and terminators occur immediately outside the coding sequence.  By convention, the gene is written " left to right " in the 5 ' to 3 ' direction of the sense strand.  The region to the ' left ' of the gene is called ' upstream ' and the region to the ' right ' is ' downstream '.

DNA to RNA – Transcription DNA transcription produces a single single-stranded RNA molecule that is complementary to one strand of DNA Molecular Biology of the Cell. 4 th edition. Poly-deoxyribonucleotide Poly-ribonucleotide

Structure of RNA Molecular Biology of the Cell. 4 th edition. RNA can fold into specific structures RNA is largely single-stranded, but it often contains short stretches of nucleotides that can form conventional base-pairs with complementary sequences and “nonconventional” base- pairs with modified bases on the same molecule that allow an RNA molecule to fold into a three-dimensional structure.

Types of RNAs  Three main types of RNAs, viz. mRNA, rRNA and tRNA are found both in prokaryotes and eukaryotes.  Transfer-messenger RNAs (tmRNA) are found only in bacteria.  Small nuclear RNAs (snRNA) are found only in nucleated organism, such as eukaryotes.  Some RNAs are involved in protein synthesis, some are in post-transcriptional modification, some are in replication and some are involved in regulation of gene expression and others.

Initiation of Transcription  The RNA polymerase recognize and binds to Pribnow box in prokaryotes, whereas a TATA box binding protein (TBP) recognize and bind to TATA box in eukaryotes).  In prokaryotes, there are no transcription factor  In eukaryotes, there are six general transcription factors namely TFIIA, TFIIB (ortholog of Archeal TFB), TFIID (a subunit ortholog of Archeal TPB), TFIIE (ortholog of archeal TFE), TFIIF and TFIIH. Closing complex in prokaryotes Closing complex in eukaryotes

Initiation of Transcription Promoter region  Region that contains specific DNA sequences recognized by a transcription factors.  Transcription factor binds to promoter sequence and recruits RNA polymerase.  It is of two types: o Core promoter: a minimal portion of the promoter required to properly initiate transcription. It has following characteristics:

• It has Transcription Start Site (TSS) upstream to the gene
• It has stretch of approximately - 34 where RNA polymerase binds
• It is a binding site for general transcription factor o Proximal promoter: is the proximal sequence upstream of the gene that tends to contain primary regulatory elements.
• It may have up to approximately - 250 bp of stretch
• It is a binding site for specific transcription factor Basal Promoter Element

Initiation of Transcription Difference between prokaryotic and eukaryotic promoters  Prokaryotic promoters: In prokaryotic, the promoter consists of two short sequences at - 10 and - 35 positions from the transcription start sites (TSS). → The sequence at - 10 is called the Pribnow box, or the - 10 element, which usually consists of six nucleotides TATAAT. It is essential to start transcription in prokaryotes. → The sequence at - 35 is called - 35 element which usually consists of the six nucleotides TTGACA. Its presence allows a very high transcription rate.  Eukaryotic promoters: In eukaryotic, the promoters are extremely diverse and are difficult to characterize. They lie upstream to gene and may have regulatory elements several kilobases away from the transcription start site. transcriptional complex can cause the DNA to bend back on itself, which allows for placement of regulatory sequences far from the actual site of transcription. → It contains a TATA box (sequence TATAAA), which binds to TATA binding protein that assists in the formation of the RNA polymerase transcriptional complex. TATA box typically lies very close to the transcription start site (often within 50 bases). http://www.scfbio-iitd.res.in/tutorial/promoter.html

Initiation of Transcription

Initiation of Transcription Transcription Cycle

Elongation of Transcripts  The RNA polymerase transcribes the DNA (β subunit initiates the synthesis), but produces about 10 abortive (short, non-productive) transcripts which are unable to leave the RNA polymerase because the exit channel is blocked by σ-factor.  The σ-factor eventually dissociates from the core enzyme and elongation proceeds.  This enzyme has no exo/endonuclease activity and cannot repair the mistakes as DNA polymerase in replication.

Elongation of Transcripts  Formation of closed complex by binding of the promoter sequence.  Thereafter, the RNA polymerase holoenzyme separates 10 - 14 bases extending from - 11 to + 3 to form a open complex called melting. The changing from closed to open complex is called isomerization.  RNA polymerase starts synthesizing RNA.  RNA polymerase exhibits groove which have length of 55 Å and diameter of 25 Å.  This groove fits well the 20 Å double strand of DNA.  The 55 Å length can accept 16 nucleotides.

Elongation of Transcripts