Lecture 5: Molecular Biology I, BIO316 PDF

Molecular Biology I BIO316 Lecture 5 Gene Expression: Transcription in prokaryotes Prepared by Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics ...

Molecular Biology I BIO316 Lecture 5 Gene Expression: Transcription in prokaryotes Prepared by Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  Dr. Rami Elshazli Structure of RNA Associate Professor of Biochemistry and Molecular Genetics  Ribozymes are catalytic RNA molecules that may have  Thymine, one of the two been the first carriers of genetic information. pyrimidines found in DNA, is  Ribozymes are RNA molecules that could catalyze replaced by uracil in RNA. specific biochemical reactions, including RNA splicing in  RNA is usually single stranded, gene expression. whereas DNA consists of two polynucleotide strands joined  Ribonucleic acid (RNA) is a polymer consisting of by hydrogen bonding. nucleotides joined together by phosphodiester bonds.  Short commentary regions of RNA can be paired and form  However, there are several important differences in secondary structures. the structures of DNA and RNA.  DNA nucleotides contain deoxyribose sugars, while RNA nucleotides have ribose sugars.  RNA is degraded rapidly under alkaline conditions due to the presence of free hydroxyl group on the 2’- carbon atom of the ribose sugar.  The deoxyribose sugar of DNA lacks this free hydroxyl group; so, DNA is a more stable molecule.  Dr. Rami Elshazli Classes of RNA Associate Professor of Biochemistry and Molecular Genetics Comparison between DNA and RNA  RNA molecules perform a variety of functions inside the Characteristics DNA RNA cell.  Ribosomal RNA (rRNA) along with ribosomal protein Composed of nucleotides Yes Yes subunits make up the ribosome, the site of protein Type of sugar Deoxyribose Ribose assembly. Presence of 2’-OH group No Yes Bases A, G, C, T A, G, C, U  Messenger RNA (mRNA) carries the coding Double or single stranded Usually double Usually single instructions from DNA to the ribosome. Secondary structure Double helix variable  After attaching to a ribosome, mRNA molecule Stability Stable Easily degraded specifies the sequence of the amino acids in a polypeptide chain.  Large precursor molecules, which are termed pre- messenger RNAs (pre-mRNAs), are the immediate products of transcription in eukaryotic cells.  Pre-mRNAs (also called primary transcripts) are modified before becoming mRNA and exiting the nucleus for translation into protein.  Dr. Rami Elshazli Classes of RNA Associate Professor of Biochemistry and Molecular Genetics  Bacterial cells do not possess pre-mRNA; transcription  Additional classes of RNA molecules are found in the  Small nucleolar RNAs takes place concurrently with translation. nuclei of eukaryotic cells. (snoRNAs) take part in  Transfer RNA (tRNA) serves as the link between the coding  Small nuclear RNAs (snRNAs) combine with small the processing of rRNA. sequence of nucleotides in the mRNA and the amino acid protein subunits to form small nuclear sequence of a polypeptide chain. ribonucleoproteins (snRNPs, known as “snurps”).  A class of very small RNA  Each tRNA attaches to one specific type of amino acid and  Function: snRNAs participate in the processing of RNA, molecules: helps to incorporate that amino acid into a polypeptide  MicroRNAs (miRNAs). converting pre-mRNA into mRNA. chain.  Small interfering RNAs (siRNAs). Location and functions of different classes of RNA molecules Class of RNA Cell type Location Function  They are found in eukaryotic cells. Ribosomal RNA (rRNA) Bacterial + Eukaryotes Cytoplasm Structural and functional components of the ribosome  They carried out RNA Messenger RNA (mRNA) Bacterial + Eukaryotes Nucleus/Cytoplasm Carries genetic code for proteins interference (RNAi), a Transfer RNA (tRNA) Bacterial + Eukaryotes Cytoplasm Helps incorporate amino acids into polypeptide chain process in which these Small nuclear RNA (snRNA) Eukaryotic Nucleus Processing of pre-mRNA small RNA molecules help trigger the Small nucleolar RNA (snoRNA) Eukaryotic Nucleus Processing and assembly of rRNA degradation of mRNA or MicroRNA (miRNA) Eukaryotic Cytoplasm Inhibits translation of mRNA inhibit their translation into protein. Small interfering RNA (siRNA) Eukaryotic Cytoplasm Triggers degradation of other RNA molecules  Dr. Rami Elshazli The concept of transcription Associate Professor of Biochemistry and Molecular Genetics All cellular RNAs are synthesized from DNA templates through the transcription process.  During replication, all the nucleotides in the DNA template are copied.  During transcription, only small parts of the DNA molecule (single gene or few genes) are transcribed into RNA.  Transcription is a highly selective process: individual genes are transcribed only as their products are needed.  Transcription requires three major components:  DNA template.  Raw materials (substrates) needed to build a new RNA molecule.  The transcription apparatus, consisting of the proteins necessary to catalyze the synthesis of RNA.  Dr. Rami Elshazli The Template for transcription Associate Professor of Biochemistry and Molecular Genetics  The template for RNA synthesis is a single strand of the DNA double helix.  Within a single gene,  Unlike replication, the transcription of a gene takes only one of the two place on only one of the two nucleotide strands of DNA strands, the DNA. template strand, is  The nucleotide strand used for transcription is termed usually transcribed the template strand. into RNA. (3’ – 5’)  The other strand, called the non-template strand (coding strand), is not ordinarily transcribed.  Dr. Rami Elshazli The Template for transcription Associate Professor of Biochemistry and Molecular Genetics Although only one strand within a single gene is normally transcribed, different genes may be transcribed from different strands. During transcription, RNA molecule that is complementary and antiparallel to the DNA template strand is synthesized. The RNA transcript has the same base sequence as that of the non-template strand, with the exception that RNA contains U rather than T.  Dr. Rami Elshazli The transcription unit Associate Professor of Biochemistry and Molecular Genetics  A transcription unit is a piece of DNA that encodes an RNA molecule.  Each transcription unit includes three regions:  Promoter.  RNA-coding region.  Terminator.  The promoter is a DNA sequence that the  The second critical region of the transcription unit is transcription apparatus recognizes and binds. the RNA-coding region, a sequence of DNA  It is located next to the transcription start site but is nucleotides that is copied into an RNA molecule. not transcribed.  The third component of the transcription unit is the  Function: terminator, a sequence of nucleotides that signals  It indicates which of the two DNA strands is to be read where transcription is to end. as the template and the direction of transcription.  Terminators are usually part of the coding sequence.  It determines the transcription start site, the first  Transcription stops only after the terminator has been nucleotide that will be transcribed into RNA. copied into RNA.  Dr. Rami Elshazli The transcription unit Associate Professor of Biochemistry and Molecular Genetics  Upstream and downstream terms:  These terms refer to the direction of transcription and the location of nucleotide sequences surrounding the RNA-coding sequence.  The transcription apparatus is said to move downstream as transcription takes place.  It binds to the promoter (which is usually upstream of the start site) and moves toward the terminator (which is downstream of the start site).  The sequence of the non-template (coding) strand will be the same as the sequence of the RNA transcribed from the template.  The exception that U in RNA replaces T in DNA.  The sequence on the non-template strand is written with the 5’ end on the left and the 3’ end on the right.  Dr. Rami Elshazli The transcription unit Associate Professor of Biochemistry and Molecular Genetics  Nomenclature around transcription start site  The first nucleotide transcribed at the transcription start site is numbered +1.  Nucleotides downstream of the start site are assigned positive numbers, and nucleotides upstream of the start site are assigned negative numbers.  There is no nucleotide assigned (0). The substrate for transcription  Nucleotides are added to the 3’ end of the RNA molecule, and the transcription is therefore 5’-3’ direction.  The synthesis of RNA is complementary and antiparallel to the DNA template strand.  Unlike DNA synthesis, RNA synthesis does not require a primer.  Dr. Rami Elshazli The transcription apparatus Associate Professor of Biochemistry and Molecular Genetics  The RNA polymerase carries out all the required steps of transcription.  The action of RNA polymerase is enhanced by various accessory proteins that join and leave the polymerase at different stages of the process.  Bacterial RNA polymerase:  Bacterial cells typically possess only one type of RNA polymerase, which catalyzes the synthesis of all  The sigma factor controls the binding of RNA classes of bacterial RNA: mRNA, tRNA, and rRNA. polymerase to the promoter.  Bacterial RNA polymerase is a large, multimeric  With sigma factor, RNA polymerase binds stably only enzyme; meaning that it consists of several to the promoter region and initiates transcription at polypeptide chains. the proper start site.  This enzyme catalyzes the elongation of the RNA  Sigma is required only for promoter binding. molecule by the addition of RNA nucleotides.  When a few RNA nucleotides have been joined  The core enzyme binds with a sigma factor (σ) to form together, sigma usually detaches from the core a holoenzyme. enzyme.  The transcription apparatus  Eukaryotic RNA polymerase:  All eukaryotic polymerases are large, multimeric enzymes, typically consisting of more than a dozen subunits.  Most eukaryotic cells possess three distinct types of RNA polymerase:  RNA polymerase I  transcribes large rRNA.  RNA polymerase II  transcribes pre-mRNAs.  In addition, some snoRNAs, miRNAs, and snRNAs.  RNA polymerase III  transcribes tRNAs and small rRNA. Eukaryotic RNA polymerase  In addition, some miRNAs, and snRNAs. Type Function RNA Polymerase I Transcribes large rRNA RNA polymerase II Transcribes pre-mRNA Some snRNAs, snoRNA, miRNAs RNA polymerase III Transcribes tRNAs, small rRNA Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics Some snRNAs, miRNAs RNA polymerase IV Transcribes some siRNAs in plants  Dr. Rami Elshazli The process of bacterial transcription Associate Professor of Biochemistry and Molecular Genetics  Transcription can be divided into three stages:  Initiation: the transcription apparatus assembles on the promoter and begins the synthesis of RNA.  Elongation: the RNA polymerase unwinding the DNA and adding new nucleotides to the 3’ end of the growing RNA strand.  Termination: the recognition of the end of the transcription unit and the separation of the RNA molecule from the DNA template.  Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  Initiation  Initiation step begins RNA synthesis with promoter recognition and formation of the transcription bubble.  Firstly, the binding of RNA polymerase to the promoter determines which parts of the DNA template are to be transcribed.  Promoters are DNA sequences that are recognized by the transcription apparatus.  In bacterial cells, promoters are usually adjacent to an RNA coding sequence.   Initiation  A consensus sequence is short stretches of nucleotides that is located within DNA in the promoter region.  The common consensus sequence found in bacterial promoters is centered about 10 bp upstream of the start site.  It is called the (–10) consensus sequence or the Pribnow box.  It is often written simply as TATAAT.  Another consensus sequence in bacterial promoters is TTGACA, which lies approximately 35 nucleotides upstream of the start site.  It is termed the –35 consensus sequence.  A promoter is a DNA sequence adjacent to a gene and required for transcription.  Promoters contain short consensus sequences that are important in the initiation of transcription. Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics   Initiation  The sigma factor associates with the core enzyme to form a holoenzyme, which binds to the –35 and –10 consensus sequences in the DNA promoter.  Although it binds only the nucleotides of consensus sequences, the enzyme extends from –50 to +20 when bound to the promoter.  After the holoenzyme has attached to the promoter, RNA polymerase is positioned over the start site for transcription (at position +1) and unwind the DNA to produce a single-stranded template.  RNA polymerase pairs the base on a ribonucleoside triphosphate with its complementary base at the start site on the DNA template strand.  No primer is required to initiate the synthesis of the RNA molecule in the 3’-end direction. Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics   Elongation  At the end of initiation, RNA polymerase undergoes a change in conformation and is thereafter no longer able to bind to the consensus sequences in the promoter.  The sigma subunit is usually released after initiation.  Transcription takes place within a short stretch of about 18 nucleotides of unwound DNA called the transcription bubble.  As it moves downstream along the template, RNA polymerase unwinds the DNA at the downstream edge of the transcription bubble and rewinds the DNA at the upstream edge of the bubble.  Transcription takes place within the transcription bubble.  DNA is unwound ahead of the bubble and rewound behind it. Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics   Termination  RNA polymerase adds nucleotides to the 3’ end of the growing RNA molecule until it transcribes a terminator.  Most terminators are found upstream of the site at which termination takes place.  Transcription therefore does not suddenly stop when polymerase reaches a terminator, as does a car stopping at a stop sign.  Transcription stops after the terminator has been transcribed, like a car that stops only after running over a speed bump.  At the terminator: RNA polymerase must stop synthesizing RNA, the RNA molecule must be released from RNA polymerase, the newly made RNA molecule must dissociate fully from the DNA, and RNA polymerase must detach from the DNA template. Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  Dr. Rami Elshazli  Termination Associate Professor of Biochemistry and Molecular Genetics  Transcription ends after RNA polymerase transcribes a terminator.  Bacterial cells possess two major types of terminators.  Rho-dependent terminators, which RNA polymerase can recognize terminators only with the help of specific protein called rho factor.  Rho-independent terminators, which RNA polymerase can recognize terminators by itself and cause the end of transcription in the absence of rho factor.  In bacteria, a group of genes is often transcribed into a single RNA molecule, which is termed a polycistronic RNA.  Polycistronic RNA is produced when a single terminator is present at the end of a group of several genes that are transcribed together, instead of each gene having its own terminator.  Polycistronic mRNA is uncommon in eukaryotes.  Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics